Note: Descriptions are shown in the official language in which they were submitted.
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METHOD, ARRAY AND USE THEREOF
Field of Invention
The present invention relates to methods for diagnosis of pancreatic cancer,
and
biomarkers and arrays for use in the same.
Background
Despite major efforts, pancreatic cancer (PaC) still carries a poor prognosis
[1]. While
PaC is only the 10th most common cancer, it is the 4th leading cause of cancer
death in
the USA [2-4]. In fact, the 5-year survival is <5%, the lowest of all
malignancies [2-3].
However, recent data have shown that the outcome could be dramatically
improved by
early detection when the cancer is still predominantly at stage I, as
illustrated by a 5-year
survival of 30-60% (5 20 mm sized tumour) and even >75% (5 10 mm sized tumour)
after
early PaC resection [2-4].
PaC is characterized by a rapid tumour progression, early metastasization, and
unresponsiveness to most conventional therapies [1, 5]. The poor prognosis is
mainly
due to the lack of effective early diagnostics combined with that disease-
specific clinical
symptoms occur late in the course of the disease. At the time of diagnosis,
the tumour
has often reached a size of 30-40 mm and a majority of all patients (52%)
already have
metastases, 26% locally advanced cancer, and only 7% have tumours confined to
the
pancreas [2, 4]. At this time, about 15% of the patients are still operable,
but their median
survival is only 20 months.
A variety of non-invasive methodologies, including (endoscopic) ultrasound,
computed
tomography, and/or endoscopic retrograde cholangio-pancreatography, are used
for PaC
diagnostics [1-2, 6]. Albeit powerful, these methods are not specific for PaC
and not
designed for early detection when the tumour is still small and potentially
curable. The
situation is further complicated by the fact that PaC is difficult to
differentiate from benign
conditions, such as chronic pancreatitis, using currently available diagnostic
tools [2].
Hence, the use of biomarkers for specific and early detection of PaC would be
of
invaluable clinical benefit.
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In spite of major efforts, molecular fingerprints associated with PaC from in
particular,
crude, non-fractionated serum and plasma, remains to be deciphered [2, 7-9].
Among the
number of mainly single biomarkers that have been outlined so far, including
e.g. CRP,
CA 242, GDF-15, haptoglobin, M2-pyruvate kinase, serum amyloid A, IGFBP-1,
none
have proven to be clinically superior to CA 19-9 [2, 8-10]. Still, the use of
CA 19-9 is
significantly hampered by the fact that it has been found to i) be elevated in
both
non-malignant conditions (e.g. pancreatitis and acute cholangitis) and other
gastro-
intestinal cancers (e.g. gastric cancer and colorectal cancer), ii) lack
sensitivity for early
PaC, and iii) be absent in about 10% of the population [2, 8-10]. When
screening for
PaC, CA 19-9 has only yielded medium sensitivity (ranging from 69% to 98%) and
specificity (46% to 98%) [2, 9-11].
Against this background, the inventors developed a proteomic approach to
prognostic
diagnosis of cancer in WO 2008/117067 whereby the first sets of serum
biomarkers for
detection of pancreatic cancer and for predicting survival were identified.
Summary of the Invention
Motivated by a recent study, in which we indicated that affinity proteomics
[12-13] could
be used to pin-point candidate PaC serum biomarker signatures [14], we have
further
deciphered the serum proteome of PaC.
In this study, we have for the first time pre-validated multiplexed serum
biomarker
signatures for PaC diagnosis, demonstrating that diagnostic information could
be
extracted from crude blood samples, displaying high specificity and
sensitivity. This
provides enhanced PaC diagnosis and thereby improved prognosis, bringing
significantly
added clinical value, as well as shedding further light on the underlying,
intricate disease
biology.
Accordingly, a first aspect of the invention provides a method for determining
the
presence of pancreatic cancer in an individual comprising or consisting of the
steps of:
a) providing a sample to be tested from the individual;
b) determining a biomarker signature of the test sample by measuring the
expression in the test sample of one or more biomarkers selected from the
group defined in Table III;
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wherein the expression in the test sample of one or more biomarkers selected
from the
group defined in Table III is indicative of the individual having pancreatic
cancer.
By "sample to be tested", "test sample" or "control sample" we include tissue,
fluid
proteome and/or expressome samples from an individual to be tested or a
control
individual, as appropriate.
By "expression" we mean the level or amount of a gene product such as mRNA or
protein.
Methods of detecting and/or measuring the concentration of protein and/or
nucleic acid
are well known to those skilled in the art, see for example Sambrook and
Russell, 2001,
Cold Spring Harbor Laboratory Press.
By "biomarker" we mean a naturally-occurring biological molecule, or component
or
fragment thereof, the measurement of which can provide information useful in
the
prognosis of pancreatic cancer. For
example, the biomarker may be a
naturally-occurring protein or carbohydrate moiety, or an antigenic component
or
fragment thereof.
In one embodiment, the method comprises or consists of steps (a) and (b) and
the
further steps of:
c) providing a control sample from an individual not afflicted with
pancreatic
cancer (i.e. a negative control);
d) determining a biomarker signature of the control sample by measuring the
expression in the control sample of the one or more biomarkers measured
in step (b);
wherein the presence of pancreatic cancer is identified in the event that the
expression in
the test sample of the one or more biomarkers measured in step (b) is
different from the
expression in the control sample of the one or more biomarkers measured in
step (d).
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In another embodiment, the method comprises or consists of steps (a), (b), (c)
and (d)
and the additional steps of:
e)
providing a control sample from an individual afflicted with pancreatic
cancer (i.e. a positive control);
determining a biomarker signature of the control sample by measuring the
expression in the control sample of the one or more biomarkers measured
in step (b);
wherein the presence of pancreatic cancer is identified in the event that the
expression in
the test sample of the one or more biomarkers measured in step (b) corresponds
to the
expression in the control sample of the one or more biomarkers measured in
step (f).
By "corresponds to the expression in the control sample" we include that the
expression
of the one or more biomarkers in the sample to be tested is the same as or
similar to the
expression of the one or more biomarkers of the positive control sample.
Preferably the
expression of the one or more biomarkers in the sample to be tested is
identical to the
expression of the one or more biomarkers of the positive control sample.
Differential expression (up-regulation or down regulation) of biomarkers, or
lack thereof,
can be determined by any suitable means known to a skilled person.
Differential
expression is determined to a p value of a least less than 0.05 (p = < 0.05),
for example,
at least <0.04, <0.03, <0.02, <0.01, <0.009, <0.005, <0.001, <0.0001, <0.00001
or at
least <0.000001. Preferably, differential expression is determined using a
support vector
machine (SVM). Preferably, the SVM is an SVM as described below. Most
preferably,
the SVM is the SVM described in Table V(A), below.
It will be appreciated by persons skilled in the art that differential
expression may relate
to a single biomarker or to multiple biomarkers considered in combination
(i.e. as a
biomarker signature). Thus, a p value may be associated with a single
biomarker or with
a group of biomarkers. Indeed, proteins having a differential expression p
value of
greater than 0.05 when considered individually may nevertheless still be
useful as
biomarkers in accordance with the invention when their expression levels are
considered
in combination with one or more other biomarkers.
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In one embodiment, step (b) comprises or consists of measuring the expression
of one
or more of the biomarkers listed in Table IV(A), for example, at least 2 of
the biomarkers
listed in Table IV(A).
As exemplified in the accompanying examples, the expression of certain
proteins in a
blood, serum or plasma test sample may be indicative of pancreatic cancer in
an
individual. For example, the relative expression of certain serum proteins in
a single test
sample may be indicative of the presence of pancreatic cancer in an
individual.
Preferably, the individual is a human. However, the individual being tested
may be any
mammal, such as a domesticated mammal (preferably of agricultural or
commercial
significance including a horse, pig, cow, sheep, dog and cat).
Preferably, step (b) comprises or consists of measuring the expression of
interleukin-7
(IL-7) and/or integrin alpha-10, for example, measuring the expression of
interleukin-7,
measuring the expression of integrin alpha-10, or measuring the expression of
interleukin-7 and integrin alpha-10.. Most preferably, step (b) comprises or
consists of
measuring the expression of each the biomarkers listed in Table IV(A).
In one embodiment, step (b) comprises or consists of measuring the expression
of 1 or
more biomarkers from the biomarkers listed in Table IV(B), for example at
least 2, 3, 4, 5,
6, 7, 8, 9, 10, 11 or 12 of the biomarkers listed in Table IV(B). Hence, step
(b) preferably
comprises or consists of measuring the expression of all of the biomarkers
listed in
Table IV(B).
In another embodiment, step (b) comprises or consists of measuring the
expression of 1
or more biomarkers from the biomarkers listed in Table IV(C), for example at
least 2, 3,
4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,
25, 26, 27, 28,
29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40 or 41 of the biomarkers listed
in Table IV(C).
Preferably, step (b) comprises or consists of measuring the expression of all
of the
biomarkers listed in Table IV(C).
Preferably, step (b) comprises or consists of measuring the expression in the
test sample
of all of the biomarkers defined in Table IV.
In one embodiment, the method is for differentiating between pancreatic cancer
(PaC)
and another disease state.
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Preferably, step (b) comprises or consists of measuring the expression in the
test sample
of 1 or more biomarkers from the biomarkers listed in Table V(A), for example
at least 2,
3, 4, 5, 6, 7, 8, 9 or 10 of the biomarkers listed in Table V(A). Preferably,
step (b) also
comprises or consists of measuring the expression in the test sample of 1 or
more
biomarkers from the biomarkers listed in Table V(B), for example at least 2,
3, 4, 5, 6, 7,
8, 9 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24 of the
biomarkers listed in
Table V(B). It is also preferred that step (b) comprises or consists of
measuring the
expression in the test sample of 1 or more biomarkers from the biomarkers
listed in
Table V(C), for example at least 2, 3, 4 or 5 of the biomarkers listed in
Table V(C).
Preferably, step (b) comprises or consists of measuring the expression in the
test sample
of 1 or more biomarkers from the biomarkers listed in Table V(D), for example
at least 2
or 3 of the biomarkers listed in Table V(D). Preferably, step (b) comprises or
consists of
measuring the expression in the test sample of 1 or more biomarkers from the
biomarkers listed in Table V(F), for example at least 2, 3, 4, 5 or 6 of the
biomarkers
listed in Table V(F). Preferably, step (b) comprises or consists of measuring
the
expression in the test sample of all of the biomarkers listed in Table V(A),
Table V(B),
Table V(C), Table V(D) and/or Table V(F).
By "differentiating between pancreatic cancer (PaC) and another disease state"
we
include differentiating between PaC and any other condition, including a state
of health.
In one embodiment, the other disease state or states is chronic pancreatitis
(ChP), acute
inflammatory pancreatitis (AIP) and/or normal, for example, the other disease
state or
states may be chronic pancreatitis alone; acute inflammatory pancreatitis
alone; chronic
pancreatitis and acute inflammatory pancreatitis; chronic pancreatitis and
normal; acute
inflammatory pancreatitis and normal; or, chronic pancreatitis, acute
inflammatory
pancreatitis and normal.
When referring to a "normal" disease state we include individuals not
afflicted with
chronic pancreatitis (ChP) or acute inflammatory pancreatitis (AIP).
Preferably the
individuals are not afflicted with any pancreatic disease or disorder. Most
preferably, the
individuals are healthy individuals, i.e., they are not afflicted with any
disease or disorder.
In another embodiment, the method is for differentiating between pancreatic
cancer and
chronic pancreatitis (ChP). Preferably, step (b) comprises or consists of
measuring the
expression in the test sample of 1 or more biomarkers from the biomarkers
listed in
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Table V(A), for example at least 2, 3, 4, 5, 6, 7, 8, 9 or 10 of the
biomarkers listed in
Table V(A). Step (b) may comprise or consist of measuring the expression in
the test
sample of 1 or more biomarkers from the biomarkers listed in Table V(C), for
example at
least 2, 3, 4 or 5 of the biomarkers listed in Table V(C). Step (b) may
comprise or consist
of measuring the expression in the test sample of all of the biomarkers listed
in Table
V(A) and/or Table V(C).
In an additional/alternative embodiment, the method is for differentiating
between
pancreatic cancer and acute inflammatory pancreatitis (AIP) and step (b)
comprises or
consists of measuring the expression in the test sample of 1 or more
biomarkers from the
biomarkers listed in Table V(A), for example at least 2, 3, 4, 5, 6, 7, 8, 9
or 10 of the
biomarkers listed in Table V(A). Preferably, step (b) comprises or consists of
measuring
the expression in the test sample of 1 or more biomarkers from the biomarkers
listed in
Table V(B), for example at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,
15, 16, 17, 18,
19, 20, 21, 22, 23 or 24 of the biomarkers listed in Table V(B). Step (b) may
comprise or
consist of measuring the expression in the test sample of 1 or more biomarkers
from the
biomarkers listed in Table V(C), for example at least 2, 3, 4 or 5 of the
biomarkers listed
in Table V(C). Preferably, step (b) comprises or consists of measuring the
expression in
the test sample of 1 or more biomarkers from the biomarkers listed in Table
V(E).
Preferably, step (b) comprises or consists of measuring the expression in the
test sample
of 1 or more biomarkers from the biomarkers listed in Table V(F), for example
at least 2,
3, 4, 5 or 6 of the biomarkers listed in Table V(F). Preferably, step (b)
comprises or
consists of measuring the expression in the test sample of 1 or more
biomarkers from the
biomarkers listed in Table V(H), for example at least 2 or 3 of the biomarkers
listed in
Table V(H). Hence, step (b) preferably comprises or consists of measuring the
expression in the test sample of all of the biomarkers listed in Table V(A),
Table V(B),
Table V(C), Table V(E), Table V(F) and/or Table IV(H).
In one embodiment, the method is for differentiating between pancreatic cancer
and
normal (N). For a definition of "normal" disease state, see above. Preferably,
step (b)
comprises or consists of measuring the expression in the test sample of 1 or
more
biomarkers from the biomarkers listed in Table V(A), for example at least 2,
3, 4, 5, 6, 7,
8, 9 or 10 of the biomarkers listed in Table V(A). Preferably, step (b)
comprises or
consists of measuring the expression in the test sample of 1 or more
biomarkers from the
biomarkers listed in Table V(B), for example at least 2, 3, 4, 5, 6, 7, 8, 9,
10, 11, 12, 13,
14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24 of the biomarkers listed in Table
V(B).
Preferably, step (b) comprises or consists of measuring the expression in the
test sample
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of 1 or more biomarkers from the biomarkers listed in Table V(D), for example
at least 2
or 3 of the biomarkers listed in Table V(D). Preferably, wherein step (b)
comprises or
consists of measuring the expression in the test sample of 1 or more
biomarkers from the
biomarkers listed in Table V(E). It is also preferred that step (b) comprises
or consists of
measuring the expression in the test sample of 1 or more biomarkers from the
biomarkers listed in Table V(G), for example at least 2 or 3 of the biomarkers
listed in
Table V(G). Hence, step (b) may comprise or consist of measuring the
expression in the
test sample of all of the biomarkers listed in Table V(A), Table V(B), Table
V(D), Table
V(E) and/or Table IV(G).
In one embodiment, step (b) comprises or consists of measuring the expression
of IL-3.
In a further embodiment, step (b) comprises or consists of measuring the
expression of
lntegrin a-10. In a still further embodiment, step (b) comprises or consists
of measuring
the expression of Mucin-1. In another embodiment, step (b) comprises or
consists of
measuring the expression of Cis. In an additional embodiment, step (b)
comprises or
consists of measuring the expression of MCP-3. In one embodiment, step (b)
comprises
or consists of measuring the expression of Angiomotin. In a further
embodiment, step (b)
comprises or consists of measuring the expression of BTK. In a still further
embodiment,
step (b) comprises or consists of measuring the expression of C1q. In another
embodiment, step (b) comprises or consists of measuring the expression of CD40
ligand.
In an additional embodiment, step (b) comprises or consists of measuring the
expression
of GM-CSF. In one embodiment, step (b) comprises or consists of measuring the
expression of IgM. In a further embodiment, step (b) comprises or consists of
measuring
the expression of IL-11. In a still further embodiment, step (b) comprises or
consists of
measuring the expression of IL-16. In another embodiment, step (b) comprises
or
consists of measuring the expression of IL-1-ra. In an additional embodiment,
step (b)
comprises or consists of measuring the expression of IL-la. In one embodiment,
step
(b) comprises or consists of measuring the expression of IL-1f3. In a further
embodiment,
step (b) comprises or consists of measuring the expression of IL-2. In a still
further
embodiment, step (b) comprises or consists of measuring the expression of IL-
7. In
another embodiment, step (b) comprises or consists of measuring the expression
of IL-9.
In an additional embodiment, step (b) comprises or consists of measuring the
expression
of INF-y. In one embodiment, step (b) comprises or consists of measuring the
expression of Integrin a-11. In a further embodiment, step (b) comprises or
consists of
measuring the expression of JAK3. In a still further embodiment, step (b)
comprises or
consists of measuring the expression of Leptin. In another embodiment, step
(b)
comprises or consists of measuring the expression of Lewis y. In an additional
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embodiment, step (b) comprises or consists of measuring the expression of MCP-
4. In
one embodiment, step (b) comprises or consists of measuring the expression of
Procathepsin W. In a further embodiment, step (b) comprises or consists of
measuring
the expression of Properdin. In a still further embodiment, step (b) comprises
or consists
of measuring the expression of PSA. In another embodiment, step (b) comprises
or
consists of measuring the expression of RANTES. In an additional embodiment,
step (b)
comprises or consists of measuring the expression of Sialyl Lewis x. In one
embodiment, step (b) comprises or consists of measuring the expression of TM
peptide.
In a further embodiment, step (b) comprises or consists of measuring the
expression of
TNF-a. In a still further embodiment, step (b) comprises or consists of
measuring the
expression of C4. In another embodiment, step (b) comprises or consists of
measuring
the expression of 6-galactosidase.
In an additional embodiment, step (b) comprises or consists of measuring the
expression
of IL-12. In one embodiment, step (b) comprises or consists of measuring the
expression of TGF-61. In a further embodiment, step (b) comprises or consists
of
measuring the expression of VEGF. In a still further embodiment, step (b)
comprises or
consists of measuring the expression of IL-8. In another embodiment, step (b)
comprises or consists of measuring the expression of C3. In an additional
embodiment,
step (b) comprises or consists of measuring the expression of IFN-y. In one
embodiment, step (b) comprises or consists of measuring the expression of IL-
10. In a
further embodiment, step (b) comprises or consists of measuring the expression
of IL-13.
In a still further embodiment, step (b) comprises or consists of measuring the
expression
of IL-18. In another embodiment, step (b) comprises or consists of measuring
the
expression of IL-6. In an additional embodiment, step (b) comprises or
consists of
measuring the expression of Lewis x. In one embodiment, step (b) comprises or
consists
of measuring the expression of Eotaxin. In a further embodiment, step (b)
comprises or
consists of measuring the expression of Cl esterase inhibitor. In a still
further
embodiment, step (b) comprises or consists of measuring the expression of MCP-
1. In
another embodiment, step (b) comprises or consists of measuring the expression
of
TNF46. In an additional embodiment, step (b) comprises or consists of
measuring the
expression of GLP-1. In one embodiment, step (b) comprises or consists of
measuring
the expression of IL-5. In a further embodiment, step (b) comprises or
consists of
measuring the expression of IL-4. In a still further embodiment, step (b)
comprises or
consists of measuring the expression of Factor B. In another embodiment, step
(b)
comprises or consists of measuring the expression of C5. In an additional
embodiment,
step (b) comprises or consists of measuring the expression of CD40.
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In one embodiment, step (b) does not comprise measuring the expression of IL-
3. In a
further embodiment, step (b) does not comprise measuring the expression of
lntegrin a-
10. In a still further embodiment, step (b) does not comprise measuring the
expression
of Mucin-1. In another embodiment, step (b) does not comprise measuring the
expression of Cis. In an additional embodiment, step (b) does not comprise
measuring
the expression of MCP-3. In one embodiment, step (b) does not comprise
measuring the
expression of Angiomotin. In a further embodiment, step (b) does not comprise
measuring the expression of BTK. In a still further embodiment, step (b) does
not
comprise measuring the expression of C1q. In another embodiment, step (b) does
not
comprise measuring the expression of CD40 ligand. In an additional embodiment,
step
(b) does not comprise measuring the expression of GM-CSF. In one embodiment,
step
(b) does not comprise measuring the expression of IgM. In a further
embodiment, step
(b) does not comprise measuring the expression of IL-11. In a still further
embodiment,
step (b) does not comprise measuring the expression of IL-16. In another
embodiment,
step (b) does not comprise measuring the expression of IL-1-ra. In an
additional
embodiment, step (b) does not comprise measuring the expression of IL-la. In
one
embodiment, step (b) does not comprise measuring the expression of IL-13. In a
further
embodiment, step (b) does not comprise measuring the expression of IL-2. In a
still
further embodiment, step (b) does not comprise measuring the expression of IL-
7. In
another embodiment, step (b) does not comprise measuring the expression of IL-
9. In
an additional embodiment, step (b) does not comprise measuring the expression
of INF-
y. In one embodiment, step (b) does not comprise measuring the expression of
lntegrin
a-11. In a further embodiment, step (b) does not comprise measuring the
expression of
JAK3. In a still further embodiment, step (b) does not comprise measuring the
expression of Leptin. In another embodiment, step (b) does not comprise
measuring the
expression of Lewis y. In an additional embodiment, step (b) does not comprise
measuring the expression of MCP-4. In one embodiment, step (b) does not
comprise
measuring the expression of Procathepsin W. In a further embodiment, step (b)
does not
comprise measuring the expression of Properdin. In a still further embodiment,
step (b)
does not comprise measuring the expression of PSA. In another embodiment, step
(b)
does not comprise measuring the expression of RANTES. In an additional
embodiment,
step (b) does not comprise measuring the expression of Sialyl Lewis x. In one
embodiment, step (b) does not comprise measuring the expression of TM peptide.
In a
further embodiment, step (b) does not comprise measuring the expression of TNF-
a. In
a still further embodiment, step (b) does not comprise measuring the
expression of C4.
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In another embodiment, step (b) does not comprise measuring the expression of
6-
galactosidase.
In an additional embodiment, step (b) does not comprise measuring the
expression of IL-
12. In one embodiment, step (b) does not comprise measuring the expression of
TGF-
61. In a further embodiment, step (b) does not comprise measuring the
expression of
VEGF. In a still further embodiment, step (b) does not comprise measuring the
expression of IL-8. In another embodiment, step (b) does not comprise
measuring the
expression of C3. In an additional embodiment, step (b) does not comprise
measuring
the expression of IFN-y. In one embodiment, step (b) does not comprise
measuring the
expression of IL-10. In a further embodiment, step (b) does not comprise
measuring the
expression of IL-13. In a still further embodiment, step (b) does not comprise
measuring
the expression of IL-18. In another embodiment, step (b) does not comprise
measuring
the expression of IL-6. In an additional embodiment, step (b) does not
comprise
measuring the expression of Lewis x. In one embodiment, step (b) does not
comprise
measuring the expression of Eotaxin. In a further embodiment, step (b) does
not
comprise measuring the expression of Cl esterase inhibitor. In a
still further
embodiment, step (b) does not comprise measuring the expression of MCP-1. In
another embodiment, step (b) does not comprise measuring the expression of TNF-
6. In
an additional embodiment, step (b) does not comprise measuring the expression
of GLP-
1. In one embodiment, step (b) does not comprise measuring the expression of
IL-5. In
a further embodiment, step (b) does not comprise measuring the expression of
IL-4. In a
still further embodiment, step (b) does not comprise measuring the expression
of Factor
B. In another embodiment, step (b) does not comprise measuring the expression
of C5.
In an additional embodiment, step (b) does not comprise measuring the
expression of
CD40.
By "TM peptide" we mean a peptide derived from a 10TM protein, to which the
scFv
antibody construct of SEQ ID NO: 1 below has specificity (wherein the CDR
sequences
are indicated by bold, italicised text):
MAEVQLLESGGGLVQPGGSLRLSCAASG FT FSSYGFHWVRQAPGKGLEINVSLISWDG
GSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARG TWFDPVVGQGTLVT
VSSGGGGSGGGGSGGGGSQSVLTQPPSASGTPGQRVTISCSGSSSN/GNNA VNWYQ
Q LPGTAPKLLIYRNNQRPSGVPDRFSGSKSGTSASLAI SGLRSEDEADYY CAAWDDSL
SWVFGGGTKLTVLG
[SEQ ID NO: 1]
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Hence, this scFy may be used or any antibody, or antigen binding fragment
thereof, that
competes with this scFy for binding to the 10TM protein. For example, the
antibody, or
antigen binding fragment thereof, may comprise the same CDRs as present in SEQ
ID
NO:1.
It will be appreciated by persons skilled in the art that such an antibody may
be produced
with an affinity tag (e.g. at the C-terminus) for purification purposes. For
example, an
affinity tag of SEQ ID NO: 2 below may be utilised:
DYKDHDGDYKDHDIDYKDDDDKAAAHHHHHH
[SEQ ID NO: 2]
In one embodiment, presence of pancreatic cancer is identified in the event
that the
expression in the test sample of IL-3, Integrina-10, Mucin-1, Cis, GLP-1R, MCP-
3,
Angiomotin, BTK, CD40 ligand, GM-CSF, IgM, IL-11, IL-16, IL-1-ra, IL-1a, IL-
113, IL-2,
IL-7, IL-9, INF-y, Integrina-11, JAK3, Leptin, Lewis y, MCP-4, Procathepsin W,
PSA,
RANTES, Sialyl Lewis x, TM peptide, TNF-a, C4, p-galactosidase, IL-12, TGF-
131, VEGF,
IL-8, C3, IFN-y, IL-10, IL-13, IL-18, IL-6, Lewis x, Eotaxin, Cl esterase
inhibitor, MCP-1,
TNF-13, GLP-1, IL-5, IL-4, Factor B, C5 and/or CD40 are up-regulated compared
to the
negative control(s) and/or corresponds to the expression of positive
control(s).
In another embodiment, presence of pancreatic cancer is identified in the
event that the
expression in the test sample of C1q and/or Properdin is down-regulated
compared to
the negative control(s) and/or corresponds to the expression of positive
control(s)
Generally, diagnosis is made with an ROC AUC of at least 0.55, for example
with an
ROC AUC of at least, 0.60, 0.65, 0.70, 0.75, 0.80, 0.85, 0.90, 0.95, 0.96,
0.97, 0.98, 0.99
or with an ROC AUC of 1.00. Preferably, diagnosis is made with an ROC AUC of
at
least 0.85, and most preferably with an ROC AUC of 1.
Typically, diagnosis is performed using a support vector machine (SVM), such
as those
available from http://cran.r-project.org/web/packages/e1071/index.html (e.g.
e1071 1.5-
24). However, any other suitable means may also be used.
Support vector machines (SVMs) are a set of related supervised learning
methods used
for classification and regression. Given a set of training examples, each
marked as
belonging to one of two categories, an SVM training algorithm builds a model
that
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13
predicts whether a new example falls into one category or the other.
Intuitively, an SVM
model is a representation of the examples as points in space, mapped so that
the
examples of the separate categories are divided by a clear gap that is as wide
as
possible. New examples are then mapped into that same space and predicted to
belong
to a category based on which side of the gap they fall on.
More formally, a support vector machine constructs a hyperplane or set of
hyperplanes in
a high or infinite dimensional space, which can be used for classification,
regression or
other tasks. Intuitively, a good separation is achieved by the hyperplane that
has the
largest distance to the nearest training datapoints of any class (so-called
functional
margin), since in general the larger the margin the lower the generalization
error of the
classifier. For more information on SVMs, see for example, Burges, 1998, Data
Mining
and Knowledge Discovery, 2:121-167.
In one embodiment of the invention, the SVM is 'trained' prior to performing
the methods
of the invention using biomarker profiles from individuals with known disease
status (for
example, individuals known to have pancreatic cancer, individuals known to
have acute
inflammatory pancreatitis, individuals known to have chronic pancreatitis or
individuals
known to be healthy). By running such training samples, the SVM is able to
learn what
biomarker profiles are associated with pancreatic cancer. Once the training
process is
complete, the SVM is then able whether or not the biomarker sample tested is
from an
individual with pancreatic cancer.
However, this training procedure can be by-passed by pre-programming the SVM
with
the necessary training parameters. For example, diagnoses can be performed
according
to the known SVM parameters using the SVM algorithm detailed in Table V, based
on
the measurement of any or all of the biomarkers listed in Table III or Table
IV.
It will be appreciated by skilled persons that suitable SVM parameters can be
determined
for any combination of the biomarkers listed in Table III or Table IV by
training an SVM
machine with the appropriate selection of data (i.e. biomarker measurements
from
individuals with known pancreatic cancer status).
Preferably, the method of the invention has an accuracy of at least 60%, for
example
61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%,
76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%,
91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% accuracy.
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Preferably, the method of the invention has a sensitivity of at least 60%, for
example
61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%,
76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%,
91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sensitivity.
Preferably, the method of the invention has a specificity of at least 60%, for
example
61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%,
76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%,
91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% specificity.
By "accuracy" we mean the proportion of correct outcomes of a method, by
"sensitivity"
we mean the proportion of all PaC positive sample that are correctly
classified as
positives, and by "specificity" we mean the proportion of all PaC negative
samples that
are correctly classified as negatives.
In one embodiment, the individual not afflicted with pancreatic cancer is not
afflicted with
pancreatic cancer (PaC), chronic pancreatitis (ChP) or acute inflammatory
pancreatitis
(AIP). More preferably, the healthy individual is not afflicted with any
pancreatic disease
or condition. Even more preferably, the individual not afflicted with
pancreatic cancer is
not afflicted with any disease or condition. Most preferably, the individual
not afflicted
with pancreatic cancer is a healthy individual. By a "healthy individual" we
include
individuals considered by a skilled person to be physically vigorous and free
from
physical disease.
However, in another embodiment the individual not afflicted with pancreatic
cancer is
afflicted with chronic pancreatitis. In still another embodiment, the
individual not afflicted
with pancreatic cancer is afflicted with acute inflammatory pancreatitis.
As previously mentioned the present method is for determining the presence of
pancreatic cancer in an individual. In one embodiment the pancreatic cancer is
selected
from the group consisting of adenocarcinoma, adenosquamous carcinoma, signet
ring
cell carcinoma, hepatoid carcinoma, colloid carcinoma, undifferentiated
carcinoma, and
undifferentiated carcinomas with osteoclast-like giant cells. Preferably, the
pancreatic
cancer is a pancreatic adenocarcinoma. More preferably, the pancreatic cancer
is
pancreatic ductal adenocarcinoma, also known as exocrine pancreatic cancer.
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In a further embodiment, step (b), (d) and/or step (f) is performed using a
first binding
agent capable of binding to the one or more biomarkers. It will be appreciated
by
persons skilled in the art that the first binding agent may comprise or
consist of a single
species with specificity for one of the protein biomarkers or a plurality of
different
species, each with specificity for a different protein biomarker.
Suitable binding agents (also referred to as binding molecules) can be
selected from a
library, based on their ability to bind a given motif, as discussed below.
At least one type of the binding agents, and more typically all of the types,
may comprise
or consist of an antibody or antigen-binding fragment of the same, or a
variant thereof.
Methods for the production and use of antibodies are well known in the art,
for example
see Antibodies: A Laboratory Manual, 1988, Harlow & Lane, Cold Spring Harbor
Press,
ISBN-13: 978-0879693145, Using Antibodies: A Laboratory Manual, 1998, Harlow &
Lane, Cold Spring Harbor Press, ISBN-13: 978-0879695446 and Making and Using
Antibodies: A Practical Handbook, 2006, Howard & Kaser, CRC Press, ISBN-13:
978-
0849335280 (the disclosures of which are incorporated herein by reference).
Thus, a fragment may contain one or more of the variable heavy (VH) or
variable light
(VL) domains. For example, the term antibody fragment includes Fab-like
molecules
(Better et al (1988) Science 240, 1041); Fv molecules (Skerra et al (1988)
Science 240,
1038); single-chain Fv (ScFv) molecules where the VH and VL partner domains
are linked
via a flexible oligopeptide (Bird et a/ (1988) Science 242, 423; Huston et al
(1988) Proc.
NatL Acad. ScL USA 85, 5879) and single domain antibodies (dAbs) comprising
isolated
V domains (Ward eta! (1989) Nature 341, 544).
The term "antibody variant" includes any synthetic antibodies, recombinant
antibodies or
antibody hybrids, such as but not limited to, a single-chain antibody molecule
produced
by phage-display of immunoglobulin light and/or heavy chain variable and/or
constant
regions, or other immunointeractive molecule capable of binding to an antigen
in an
immunoassay format that is known to those skilled in the art.
A general review of the techniques involved in the synthesis of antibody
fragments which
retain their specific binding sites is to be found in Winter & Milstein (1991)
Nature 349,
293-299.
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Molecular libraries such as antibody libraries (Clackson eta!, 1991, Nature
352, 624-628;
Marks et al, 1991, J Mol Biol 222(3): 581-97), peptide libraries (Smith, 1985,
Science
228(4705): 1315-7), expressed cDNA libraries (Santi et al (2000) J Mol Biol
296(2): 497-
508), libraries on other scaffolds than the antibody framework such as
affibodies
(Gunneriusson et al, 1999, App! Environ Microbiol 65(9): 4134-40) or libraries
based on
aptamers (Kenan et al, 1999, Methods Mol Biol 118, 217-31) may be used as a
source
from which binding molecules that are specific for a given motif are selected
for use in
the methods of the invention.
The molecular libraries may be expressed in vivo in prokaryotic (Clackson et
al, 1991,
op. cit.; Marks eta!, 1991, op. cit.) or eukaryotic cells (Kieke eta!, 1999,
Proc Nat! Acad
Sci USA, 96(10):5651-6) or may be expressed in vitro without involvement of
cells
(Hanes & Pluckthun, 1997, Proc Nat! Acad Sci USA 94(10):4937-42; He & Taussig,
1997, Nucleic Acids Res 25(24):5132-4; Nemoto et al, 1997, FEBS Lett,
414(2):405-8).
In cases when protein based libraries are used often the genes encoding the
libraries of
potential binding molecules are packaged in viruses and the potential binding
molecule is
displayed at the surface of the virus (Clackson et al, 1991, op. cit.; Marks
eta!, 1991, op.
cit; Smith, 1985, op. cit.).
The most commonly used such system today is filamentous bacteriophage
displaying
antibody fragments at their surfaces, the antibody fragments being expressed
as a fusion
to the minor coat protein of the bacteriophage (Clackson et al, 1991, op.
cit.; Marks et al,
1991, op. cit). However, also other systems for display using other viruses
(EP 39578),
bacteria (Gunneriusson et al, 1999, op. cit.; Daugherty et al, 1998, Protein
Eng
11(9):825-32; Daugherty et a/, 1999, Protein Eng 12(7):613-21), and yeast
(Shusta et al,
1999, J Mol Biol 292(5):949-56) have been used.
In addition, display systems have been developed utilising linkage of the
polypeptide
product to its encoding mRNA in so called ribosome display systems (Hanes &
Pluckthun, 1997, op. cit.; He & Taussig, 1997, op. cit.; Nemoto et al, 1997,
op. cit.), or
alternatively linkage of the polypeptide product to the encoding DNA (see US
Patent No.
5,856,090 and WO 98/37186).
When potential binding molecules are selected from libraries one or a few
selector
peptides having defined motifs are usually employed. Amino acid residues that
provide
structure, decreasing flexibility in the peptide or charged, polar or
hydrophobic side
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chains allowing interaction with the binding molecule may be used in the
design of motifs
for selector peptides.
For example:
(I) Proline may stabilise a peptide structure as its side chain is bound
both to the
alpha carbon as well as the nitrogen;
(ii) Phenylalanine, tyrosine and tryptophan have aromatic side chains and
are highly
hydrophobic, whereas leucine and isoleucine have aliphatic side chains and are
also hydrophobic;
(iii) Lysine, arginine and histidine have basic side chains and will be
positively
charged at neutral pH, whereas aspartate and glutamate have acidic side chains
and will be negatively charged at neutral pH;
(iv) Asparagine and glutamine are neutral at neutral pH but contain a amide
group
which may participate in hydrogen bonds;
(v) Serine, threonine and tyrosine side chains contain hydroxyl groups,
which may
participate in hydrogen bonds.
Typically, selection of binding agents may involve the use of array
technologies and
systems to analyse binding to spots corresponding to types of binding
molecules.
In one embodiment, the first binding agent(s) is/are immobilised on a surface
(e.g. on a
multiwell plate or array).
The variable heavy (VH) and variable light (VL) domains of the antibody are
involved in
antigen recognition, a fact first recognised by early protease digestion
experiments. Further
confirmation was found by "humanisation" of rodent antibodies. Variable
domains of rodent
origin may be fused to constant domains of human origin such that the
resultant antibody
retains the antigenic specificity of the rodent parented antibody (Morrison et
al (1984) Proc.
Natl. Acad. Sci. USA 81, 6851-6855).
That antigenic specificity is conferred by variable domains and is independent
of the
constant domains is known from experiments involving the bacterial expression
of antibody
fragments, all containing one or more variable domains. These molecules
include Fab-like
molecules (Better et al (1988) Science 240, 1041); Fv molecules (Skerra et al
(1988)
Science 240, 1038); single-chain Fv (ScFv) molecules where the VH and VL
partner
domains are linked via a flexible oligopeptide (Bird et al (1988) Science 242,
423; Huston et
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al (1988) Proc. Natl. Acad. Sci. USA 85, 5879) and single domain antibodies
(dAbs)
comprising isolated V domains (Ward et al (1989) Nature 341, 544). A general
review of
the techniques involved in the synthesis of antibody fragments which retain
their specific
binding sites is to be found in Winter & Milstein (1991) Nature 349, 293-299.
By "ScFv molecules" we mean molecules wherein the VH and VL partner domains
are linked
via a flexible oligopeptide.
The advantages of using antibody fragments, rather than whole antibodies, are
several-fold.
The smaller size of the fragments may lead to improved pharmacological
properties, such
as better penetration of solid tissue. Effector functions of whole antibodies,
such as
complement binding, are removed. Fab, Fv, ScFv and dAb antibody fragments can
all be
expressed in and secreted from E. coil, thus allowing the facile production of
large amounts
of the said fragments.
Whole antibodies, and F(a131)2 fragments are "bivalent". By "bivalent" we mean
that the said
antibodies and F(ab')2 fragments have two antigen combining sites. In
contrast, Fab, Fv,
ScFv and dAb fragments are monovalent, having only one antigen combining
sites.
The antibodies may be monoclonal or polyclonal. Suitable monoclonal antibodies
may
be prepared by known techniques, for example those disclosed in "Monoclonal
Antibodies: A manual of techniques", H Zola (CRC Press, 1988) and in
"Monoclonal
Hybridoma Antibodies: Techniques and applications", J G R Hurrell (CRC Press,
1982),
both of which are incorporated herein by reference.
In one embodiment, the first binding agent immobilised on a surface (e.g. on a
multiwell
plate or array).
The variable heavy (VH) and variable light (VL) domains of the antibody are
involved in
antigen recognition, a fact first recognised by early protease digestion
experiments. Further
confirmation was found by "humanisation" of rodent antibodies. Variable
domains of rodent
origin may be fused to constant domains of human origin such that the
resultant antibody
retains the antigenic specificity of the rodent parented antibody (Morrison et
al (1984) Proc.
Natl. Acad. Sci. USA 81, 6851-6855).
That antigenic specificity is conferred by variable domains and is independent
of the
constant domains is known from experiments involving the bacterial expression
of antibody
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fragments, all containing one or more variable domains. These molecules
include Fab-like
molecules (Better et a/ (1988) Science 240, 1041); Fv molecules (Skerra et a/
(1988)
Science 240, 1038); single-chain Fv (ScFv) molecules where the VH and VL
partner
domains are linked via a flexible oligopeptide (Bird et al (1988) Science 242,
423; Huston et
al (1988) Proc. Natl. Acad. Sci. USA 85, 5879) and single domain antibodies
(dAbs)
comprising isolated V domains (Ward et a/ (1989) Nature 341, 544). A general
review of
the techniques involved in the synthesis of antibody fragments which retain
their specific
binding sites is to be found in Winter & Milstein (1991) Nature 349, 293-299.
By "ScFv molecules" we mean molecules wherein the VH and VL partner domains
are linked
via a flexible oligopeptide.
The advantages of using antibody fragments, rather than whole antibodies, are
several-fold.
The smaller size of the fragments may lead to improved pharmacological
properties, such
as better penetration of solid tissue. Effector functions of whole antibodies,
such as
complement binding, are removed. Fab, Fv, ScFv and dAb antibody fragments can
all be
expressed in and secreted from E. coli, thus allowing the facile production of
large amounts
of the said fragments.
Whole antibodies, and F(ab1)2 fragments are "bivalent". By "bivalent" we mean
that the said
antibodies and F(a131)2 fragments have two antigen combining sites. In
contrast, Fab, Fv,
ScFv and dAb fragments are monovalent, having only one antigen combining
sites.
The antibodies may be monoclonal or polyclonal. Suitable monoclonal antibodies
may
be prepared by known techniques, for example those disclosed in "Monoclonal
Antibodies: A manual of techniques", H Zola (CRC Press, 1988) and in
"Monoclonal
Hybridoma Antibodies: Techniques and applications", J G R Hurrell (CRC Press,
1982),
both of which are incorporated herein by reference.
Hence, the first binding agent may comprise or consist of an antibody or an
antigen-binding fragment thereof. Preferably, the antibody or antigen-binding
fragment
thereof is a recombinant antibody or antigen-binding fragment thereof. The
antibody or
antigen-binding fragment thereof may be selected from the group consisting of:
scFv,
Fab, and a binding domain of an immunoglobulin molecule.
Preferably, the first binding agent is immobilised on a surface.
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The one or more biomarkers in the test sample may be labelled with a
detectable moiety.
By a "detectable moiety" we include the meaning that the moiety is one which
may be
detected and the relative amount and/or location of the moiety (for example,
the location
on an array) determined.
Suitable detectable moieties are well known in the art.
Thus, the detectable moiety may be a fluorescent and/or luminescent and/or
chemiluminescent moiety which, when exposed to specific conditions, may be
detected.
For example, a fluorescent moiety may need to be exposed to radiation (i.e.
light) at a
specific wavelength and intensity to cause excitation of the fluorescent
moiety, thereby
enabling it to emit detectable fluorescence at a specific wavelength that may
be
detected.
Alternatively, the detectable moiety may be an enzyme which is capable of
converting a
(preferably undetectable) substrate into a detectable product that can be
visualised
and/or detected. Examples of suitable enzymes are discussed in more detail
below in
relation to, for example, ELISA assays.
Alternatively, the detectable moiety may be a radioactive atom which is useful
in imaging.
Suitable radioactive atoms include 99mTc and 1231 for scintigraphic studies.
Other readily
detectable moieties include, for example, spin labels for magnetic resonance
imaging
(MRI) such as 1231 again, 1311, iiiin, 19F, 13C, 15N, 170, gadolinium,
manganese or iron.
Clearly, the agent to be detected (such as, for example, the one or more
biomarkers in
the test sample and/or control sample described herein and/or an antibody
molecule for
use in detecting a selected protein) must have sufficient of the appropriate
atomic
isotopes in order for the detectable moiety to be readily detectable.
The radio- or other labels may be incorporated into the agents of the
invention (i.e. the
proteins present in the samples of the methods of the invention and/or the
binding agents
of the invention) in known ways. For example, if the binding moiety is a
polypeptide it
may be biosynthesised or may be synthesised by chemical amino acid synthesis
using
suitable amino acid precursors involving, for example, fluorine-19 in place of
hydrogen.
Labels such as 99mTc, 1231, 186Rh, 188Rh and 111In can, for example, be
attached via
cysteine residues in the binding moiety. Yttrium-90 can be attached via a
lysine residue.
The IODOGEN method (Fraker et al (1978) Biochem. Biophys. Res. Comm. 80, 49-
57)
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can be used to incorporate 1231.
Reference ("Monoclonal Antibodies in
Immunoscintigraphy", J-F Chatal, CRC Press, 1989) describes other methods in
detail.
Methods for conjugating other detectable moieties (such as enzymatic,
fluorescent,
luminescent, chemiluminescent or radioactive moieties) to proteins are well
known in the
art.
Preferably, the one or more biomarkers in the control sample(s) are labelled
with a
detectable moiety. The detectable moiety may be selected from the group
consisting of:
a fluorescent moiety; a luminescent moiety; a chemiluminescent moiety; a
radioactive
moiety; an enzymatic moiety. However, it is preferred that the detectable
moiety is
biotin.
In an additional embodiment step (b), (d) and/or step (f) is performed using
an assay
comprising a second binding agent capable of binding to the one or more
biomarkers,
the second binding agent comprising a detectable moiety. Preferably, the
second
binding agent comprises or consists of an antibody or an antigen-binding
fragment
thereof. Preferably, the antibody or antigen-binding fragment thereof is a
recombinant
antibody or antigen-binding fragment thereof. Most preferably, the antibody or
antigen-
binding fragment thereof is selected from the group consisting of: scFv, Fab
and a
binding domain of an immunoglobulin molecule. In one embodiment the detectable
moiety is selected from the group consisting of: a fluorescent moiety; a
luminescent
moiety; a chemiluminescent moiety; a radioactive moiety and an enzymatic
moiety.
Preferably, the detectable moiety is fluorescent moiety (for example an Alexa
Fluor dye,
e.g. A1exa647).
In one embodiment, the method of the first aspect of the invention comprises
or consists
of an ELISA (Enzyme Linked Immunosorbent Assay).
Preferred assays for detecting serum or plasma proteins include enzyme linked
immunosorbent assays (ELISA), radioimmunoassay (RIA), immunoradiometric assays
(IRMA) and immunoenzymatic assays (IEMA), including sandwich assays using
monoclonal and/or polyclonal antibodies. Exemplary sandwich assays are
described by
David et al in US Patent Nos. 4,376,110 and 4,486,530, hereby incorporated by
reference. Antibody staining of cells on slides may be used in methods well
known in
cytology laboratory diagnostic tests, as well known to those skilled in the
art.
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Typically, the assay is an ELISA (Enzyme Linked lmmunosorbent Assay) which
typically
involves the use of enzymes giving a coloured reaction product, usually in
solid phase
assays. Enzymes such as horseradish peroxidase and phosphatase have been
widely
employed. A way of amplifying the phosphatase reaction is to use NADP as a
substrate
to generate NAD which now acts as a coenzyme for a second enzyme system.
Pyrophosphatase from Escherichia coli provides a good conjugate because the
enzyme
is not present in tissues, is stable and gives a good reaction colour. Chemi-
luminescent
systems based on enzymes such as luciferase can also be used.
ELISA methods are well known in the art, for example see The ELISA Guidebook
(Methods in Molecular Biology), 2000, Crowther, Humana Press, ISBN-13: 978-
0896037281 (the disclosures of which are incorporated by reference).
Conjugation with the vitamin biotin is frequently used since this can readily
be detected
by its reaction with enzyme-linked avidin or streptavidin to which it binds
with great
specificity and affinity.
However, step (b), (d) and/or step (f) is alternatively performed using an
array. Arrays
per se are well known in the art. Typically they are formed of a linear or two-
dimensional
structure having spaced apart (i.e. discrete) regions ("spots"), each having a
finite area,
formed on the surface of a solid support. An array can also be a bead
structure where
each bead can be identified by a molecular code or colour code or identified
in a
continuous flow. Analysis can also be performed sequentially where the sample
is
passed over a series of spots each adsorbing the class of molecules from the
solution.
The solid support is typically glass or a polymer, the most commonly used
polymers
being cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride or
polypropylene.
The solid supports may be in the form of tubes, beads, discs, silicon chips,
microplates,
polyvinylidene difluoride (PVDF) membrane, nitrocellulose membrane, nylon
membrane,
other porous membrane, non-porous membrane (e.g. plastic, polymer, perspex,
silicon,
amongst others), a plurality of polymeric pins, or a plurality of microtitre
wells, or any
other surface suitable for immobilising proteins, polynucleotides and other
suitable
molecules and/or conducting an immunoassay. The binding processes are well
known in
the art and generally consist of cross-linking covalently binding or
physically adsorbing a
protein molecule, polynucleotide or the like to the solid support. By using
well-known
techniques, such as contact or non-contact printing, masking or
photolithography, the
location of each spot can be defined. For reviews see Jenkins, R.E.,
Pennington, S.R.
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23
(2001, Proteomics, 2,13-29) and Lal et al (2002, Drug Discov Today 15;7(18
Suppl):S143-9).
Typically the array is a microarray. By "microarray" we include the meaning of
an array
of regions having a density of discrete regions of at least about 100/cm2, and
preferably
at least about 1000/cm2. The regions in a microarray have typical dimensions,
e.g.,
diameters, in the range of between about 10-250 Am, and are separated from
other
regions in the array by about the same distance. The array may also be a
macroarray or
a nanoarray.
Once suitable binding molecules (discussed above) have been identified and
isolated,
the skilled person can manufacture an array using methods well known in the
art of
molecular biology.
Hence, the array may be the array is a bead-based array or a surface-based
array.
Preferably, the array is selected from the group consisting of: macroarray,
microarray
and nanoarray.
In one embodiment, the method according to the first aspect of the invention
comprises:
(i) labelling biomarkers present in the sample with biotin;
(ii) contacting the biotin-labelled proteins with an array comprising a
plurality
of scFv immobilised at discrete locations on its surface, the scFv having
specificity for one or more of the proteins in Table III;
(iii) contacting the immobilised scFv with a streptavidin conjugate
comprising a
fluorescent dye; and
(iv) detecting the presence of the dye at discrete locations on the array
surface
wherein the expression of the dye on the array surface is indicative of the
expression of a
biomarker from Table III in the sample.
In an alternative embodiment step (b), (d) and/or (f) comprises measuring the
expression
of a nucleic acid molecule encoding the one or more biomarkers. Preferably the
nucleic
acid molecule is a cDNA molecule or an mRNA molecule. Most preferably the
nucleic
acid molecule is an mRNA molecule.
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Hence the expression of the one or more biomarker(s) in step (b), (d) and/or
(f) may be
performed using a method selected from the group consisting of Southern
hybridisation,
Northern hybridisation, polymerase chain reaction (PCR), reverse transcriptase
PCR
(RT-PCR), quantitative real-time PCR (qRT-PCR), nanoarray, microarray,
macroarray,
autoradiography and in situ hybridisation. Preferably, the expression of the
one or more
biomarker(s) in step (b) is determined using a DNA microarray.
In one embodiment, the measuring of the expression of the one or more
biomarker(s) in
step (b), (d) and/or (f) is performed using one or more binding moieties, each
individually
capable of binding selectively to a nucleic acid molecule encoding one of the
biomarkers
identified in Table III.
In a further embodiment, the one or more binding moieties each comprise or
consist of a
nucleic acid molecule. Thus, the one or more binding moieties may each
comprise or
consist of DNA, RNA, PNA, LNA, GNA, TNA or PM0. However, it is preferred that
the
one or more binding moieties each comprise or consist of DNA.
Preferably, the one or more binding moieties are 5 to 100 nucleotides in
length. More
preferably, the one or more nucleic acid molecules are 15 to 35 nucleotides in
length.
More preferably still, the binding moiety comprises a detectable moiety.
In an additional embodiment, the detectable moiety is selected from the group
consisting
of: a fluorescent moiety; a luminescent moiety; a chemiluminescent moiety; a
radioactive
moiety (for example, a radioactive atom); and an enzymatic moiety. Preferably,
the
detectable moiety comprises or consists of a radioactive atom. The radioactive
atom
may be selected from the group consisting of technetium-99m, iodine-123,
iodine-125,
iodine-131, indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17,
phosphorus-32,
sulphur-35, deuterium, tritium, rhenium-186, rhenium-188 and yttrium-90.
However, the detectable moiety of the binding moiety may be a fluorescent
moiety (for
example an Alexa Fluor dye, e.g. A1exa647).
In one embodiment the sample provided in step (b), (d) and/or (f) is selected
from the
group consisting of unfractionated blood, plasma, serum, tissue fluid,
pancreatic tissue,
pancreatic juice, bile and urine. Preferably, the sample provided in step (b),
(d) and/or (f)
is selected from the group consisting of unfractionated blood, plasma and
serum. More
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preferably, the sample provided in step (b), (d) and/or (f) is plasma. In
another preferred
embodiment, the sample provided in step (b), (d) and/or (f) is serum.
A second aspect of the present invention provides an array for determining the
presence
of pancreatic cancer in an individual comprising one or more binding agent as
defined in
the first aspect of the present invention.
Arrays suitable for use in the methods of the invention are discussed above.
Preferably the one or more binding agents are capable of binding to all of the
proteins
defined in Table III.
A third aspect of the present invention provides the use of one or more
biomarkers
selected from the group defined in the first aspect of the invention as a
diagnostic marker
for determining the presence of pancreatic cancer in an individual.
Preferably, all of the
proteins defined in Table III are used as diagnostic markers for determining
the presence
of pancreatic cancer in an individual.
A fourth aspect of the present invention provides a kit for determining the
presence of
pancreatic cancer comprising:
A) one or more first binding agent according to the first aspect of the
invention or
an array according the second aspect of the invention; and
B) instructions for performing the method according to the first aspect of the
invention .
Preferred, non-limiting examples which embody certain aspects of the invention
will now
be described, with reference to the following tables and figures:
Figure 1: Classification of PaC vs. N
A) Scanned image of an antibody microarray hybridized with a PaC serum. In
total, 160
probes, including position markers and controls, were printed in eight 20x8
subarrays per
slide. B) Differentially expressed (p<0.05) serum analytes for PaC vs. N. C)
ROC curve
for PaC vs. N based on all antibodies, i.e. using unfiltered data. D)
Classification of PaC
vs. N, using the SVM prediction values based on all antibodies (red dots-PaC,
blue dots-
N). The relative expression levels of the top 20 differentially expressed
(p<0.02) non-
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redundant analytes are shown in a heatmap. Red ¨ up-regulated, green ¨ down-
regulated, black ¨ equal levels. (E) Validation of scFv antibody specificity,
illustrated for a
highly differentially expressed (p=0.005) analyte, IL-6, using a 278 human
protein array.
(F) Validation of scFv antibody specificity, illustrated for a modestly
differentially
expressed (p=0.04) analyte, IL-10, using a 278 human protein array.
Figure 2: Pre-validation of biomarker signature for PaC vs. N classification
(A) Condensation of the biomarker signature for PaC vs. N classification in
the first
patient cohort using a LOO procedure combined with a backward elimination
strategy.
The observed ROC AUC values were plotted against the remaining number of
antibodies. (C) The condensed 18-analyte non-redundant biomarker signature
obtained
from the first patient cohort. (D) The first patient cohort was used as
training set, and the
output classifier was then tested on a new, independent patient group, the
second
patient cohort. (E) Pre-validation of the biomarker signature for PaC vs. N
classification
illustrated by the ROC curve obtained for the classifier on the test set.
Figure 3: Candidate serum biomarker signatures differentiating PaC and
pancreatitis
(A) Differentially expressed (p<0.05) serum analytes for PaC vs. ChP, AIP or
ChP+AIP+N, respectively. (B) ROC curves for PaC vs ChP, AIP, or ChP+AIP+N
classification based on all antibodies, i.e. using unfiltered data. (D)
Validation of the
antibody microarray data of selected analytes using a 10-plex cytokine
sandwich
antibody microarray (MSD). Data is only shown for the only analyte, IL-8, for
which the
majority of the observed signals were above the lower limit of detection for
the MSD
assay.
Figure 4: Pre-validation of a candidate serum biomarker signature for PaC
diagnosis
(A) The first patient cohort, composed of PaC, N, ChP and AIP, was split into
a training
set (two thirds) and a test set (one third). (B) The condensed 25 non-
redundant serum
biomarker signature obtained for the training set using a backward elimination
strategy.
(C) Pre-validation of the condensed 25-analyte biomarker signature for PaC
diagnosis,
as illustrated by the ROC curve obtained for the classifier on the test set.
(D)
Performance, expressed as ROC AUC values, of the condensed biomarker signature
obtained by the backward elimination strategy (solid line) as compared to that
of 25-
analyte signatures obtained by either i) 1000 random 25-marker signatures
(open
circles), ii) lowest p-values (dashed line), or iii) highest fold-change
(dotted line). (E)
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Comparison of the ROC AUC value obtained for the condensed 25-analyte
biomarker
signature on the test set, when the sample annotation was correct (solid line)
or
permutated a 1000 times (open circles).
Figure 5: Schematic outline of the antibody microarray strategy
Figure 6: ROC-AUC values for differentiation between (A) pancreatic cancer,
and
(B) normal, chronic pancreatitis, and/or acute inflammatory pancreatitis
ROC-AUC values are shown for marker signatures having all of the Table IV(A)
(i.e.,
core) and Table(B) (i.e., preferred) markers, and increasing numbers of
Table(C) (i.e.,
optional) markers. The best ROC AUC value (0.90) is obtained for a 29 analyte
signature, i.e., core markers + preferred markers +15 optional markers.
However, all
marker combinations had substantial predictive power.
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EXAMPLES
Materials and methods
Serum samples
Serum samples were collected at the time of diagnosis, i.e. prior to
commencing any
therapy, from two independent patient cohorts and stored at -80 C. In the
first cohort,
serum samples from 103 patients, diagnosed with pancreatic cancer (PaC)
(n=34),
chronic pancreatitis (ChP) (n=16), autoimmune pancreatitis (AIP) (n=23), or
healthy
individuals (N) (n=30) (no clinical symptoms) were screened. The patient
demographics
are described in Table 1. This cohort was randomly split and used as training
set and
test set. The second cohort, comprised of 45 patients, diagnosed with PaC
(n=25), or N
(n=20) (for patient demographics, see [14]), was used as an independent test
set only,
using antibody microarray data recently collected [14]. The size of the sample
cohorts
was limited by the availability of well-characterized serum samples collected
at the time
of diagnosis.
Antibody microarray analysis
The recombinant antibody microarray analysis was performed using previously in-
house
optimized protocols [12-15] (see below). Briefly, 121 human recombinant single-
chain Fv
(scFv) antibodies, targeting 57 mainly immunoregulatory analytes, were used as
probes.
The specificity, affinity (nM range), and on-chip functionality of the phage-
display derived
scFvs [16] was ensured by using i) stringent selection protocols [16], ii)
multiple clones
(4) per target analyte, and iii) a scFv library microarray adapted by
molecular design
(REF). The planar antibody microarrays (array size; 160x8, <0.5 cm2) were
prepared by
dispensing the antibodies and controls one-by-one (330 pUdrop) using a non-
contact
dispenser. The biotinylated serum samples were separately screened and
specifically
bound analytes were visualized by adding fluorescently labelled streptavidin
using a
confocal fluorescence scanner. Each individual array data point represents the
mean
value of four replicates. Chip-to-chip normalization was performed by using a
semi-global
normalization approach. In accordance to previous studies [12, 15, 17], the
correlation
coefficient for spot-to-spot reproducibility and array-to-array
reproducibility was 0.99 and
0.94, respectively. Selected antibody specificities and microarray data were
validated
(Table II) using a 234 human protein array and a 10-plex cytokine sandwich
antibody
microarray, respectively. In addition, several antibody specificities have
previously been
validated using ELISA, protein arrays, blocking/spiking experiments, and/or
mass
spectrometry (Table II).
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Microarray data analysis
The data analysis was performed in R (see below). Briefly, a support vector
machine
(SVM) was employed to classify the samples as belonging to one of two defined
groups
(e.g. cancer vs. healthy), using a linear kernel with the cost of constraints
set to 1. No
attempts were made to tune it in order to avoid the risk of over-fitting. The
SVM was
trained and tested using a leave-one-out (L00) cross validation procedure. In
two of the
comparisons, this training part included the creation of an antibody sub-panel
by
selecting antibodies that, in the training set, displayed the highest
discriminatory power.
This selection of antibodies was made using either a direct or a cross-
validated
backward elimination strategy. Using this approach, condensed candidate
biomarker
signatures were identified, and subsequently evaluated on independent test
sets.
Sensitivity and specificity values were calculated from the SVM decision
values, using a
threshold level of zero. A receiver operating characteristics (ROC) curve was
constructed
using the SVM decision values. The area under the curve (AUC) was calculated
and
used as a measure of prediction performance. Further, the Wilcoxon p-value and
the fold
change were calculated for each antibody. The candidate biomarker signatures
were
reported following the recommendations for tumour marker prognostic studies
[18].
Serum samples
After informed consent, serum samples were collected from two independent
patient
cohorts at the time of diagnosis, i.e. prior to initiation of therapy, and
stored at -80 C. PC
was verified with histology. Patient cohort 1 was composed of serum samples
from 103
patients (Mannheim University Hospital, Germany), diagnosed with pancreatic
ductal
adenocarcinoma (PaC) (n=34), chronic pancreatitis (hCP) (n=16), autoimmune
pancreatitis (AIP) (n=23), or healthy individuals (controls; N) (n=30) (no
clinical
symptoms). The patient demographics are described in Table 1. This cohort was
also
randomly split and used as training set (two thirds of the samples) and test
set (one
third). Patient cohort 2 was composed of 45 patients, diagnosed with PaC
(n=25), or N
(n=20) (Stockholm South General Hospital and Lund University Hospital, Sweden)
(for
patient demographics, see [39]), and was adopted using antibody microarray
data as
recently described [39]. A power analysis (see below) was performed in order
to confirm
that the size of the sample cohorts was sufficient to provide a statistical
power >80%.
The main experiments were performed on patient cohort 1, while cohort 2 was
used as
an independent data set for validation in one experiment (see Figure 5).
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Labelling of serum samples
The serum samples were labelled using previously optimized labelling protocols
for
serum proteomes [39-43]. Briefly, crude serum samples were thawed on ice and
30 pL
aliquots were centrifuged (16 000 x g for 20 min at 4 C). Five pL of the
supernatant was
diluted 45 times in PBS, resulting in a protein concentration of approximately
2 mg/mL.
Samples were labelled with EZ-link Sulfo-NHS-LC-Biotin (Pierce, Rockford, IL,
USA) at
a final concentration of 0.6 mM for 2h on ice with gently vortexing every 20
min. Free
biotin was removed by dialysis against PBS for 72 h at 4 C using 3.5 kDa MW
cut-off
dialysis units (Thermo Scientific, Rockford, IL, USA). The samples were
aliquoted and
stored at -20 C.
Production and purification of scFv
In total, 121 human recombinant single-chain Fv (scFv) antibody fragments,
targeting 57
mainly immunoregulatory biomolecules were selected from the n-CoDeR library
[43] and
kindly provided by Biolnvent International AB, Lund, Sweden, or provided by
Prof. Mats
Ohlin (Lund University, Sweden) (5 clones against mucin-1). The specificity,
affinity (nM
range) and on-chip functionality of the phage-display derived scFv was ensured
by using
i) stringent selection protocols [43], ii) multiple clones (4) per target
molecule, and iii) a
scFv library microarray adapted by molecular design [44, 45]. The antibody
fragments
were produced in 100 mL E. coli cultures and purified from either expression
supernatants or cell periplasm, using affinity chromatography on Ni-NTA
agarose
(Qiagen, Hi!den, Germany). Bound molecules were eluted with 250 mM imidazole,
extensively dialysed against PBS and stored at 4 C until used for microarray
fabrication.
The antibody concentration was determined by measuring the absorbance at 280
nm
(average 500 pg/mL, range 50¨ 1840 pg/mL).
Fabrication and processing of antibody microarrays
For the production of planar antibody microarrays, we used a set-up previously
optimized
and validated [39-43, 46]. Briefly, scFvs were arrayed onto balck polymer
Maxisorb
microarray slides (NUNC, Roskilde, Denmark) using a non-contact printer
(BioChip
Arrayer, PerkinElmer Life & Analytical Sciences, Wellesley, MA, USA) by
depositing
approximately 330 pL drops, using piezo technology. Two drops were spotted in
each
position, allowing the first drop to dry out before the second drop was
dispensed. In
average, 5 fmol antibody (rang 1.5-25) was deposited per position. In order to
ensure
adequate statistics and to account for any local defects, each probe was
printed in eight
replicates. In total, 160 probes, including position markers and control scFvs
were printed
per slide, oriented in eight 20 x 8 subarrays. To assist grid alignment during
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quantification, a row of Alexa647 conjugated Streptavidin (Invitrogen,
Carlsband, CA,
USA) (10 pg/mL) was spotted at selected positions. The arrays were blocked in
5% (w/v)
fat-free milk powder (Semper AB, Sundbyberg, Sweden) in PBS over night.
The microarray slides were processed in a ProteinArray Workstation
(PerkinElmer Life &
Analytical Sciences) according to a previously described protocol [42].
Briefly, the arrays
were washed with 0.5% (v/v) Tween-20 in PBS (PBS-T) for 4 min at 60 pL/min and
then
incubated with 75 pL biotinylated serum sample (diluted 1:2, resulting in a
total serum
dilution of 1:90) in 1% (w/v) fat-free milk powder and 1% (v/v) Tween-20 in
PBS (PBS-
MT), for 1h with agitation every 15th second. Next, the arrays were again
washed with
PBS-T and incubated with 1 pg/mL Alexa-647 conjugated streptavidin in PBS-MT,
for lh.
Finally, the arrays were washed with PBS-T, dried under a stream of nitrogen
gas and
scanned with a confocal microarray scanner (PerkinElmer Life & Analytical
Sciences) at
pm resolution, using four different scanner settings of PMT gain and laser
power. The
intensity of each spot was quantified in the ScanArray Express software v.4.0
(PerkinElmer Life & Analytical Sciences), using the fixed circle method. The
local
background was subtracted. To compensate for any possible local defects, the
two
highest and the two lowest replicates were automatically excluded and the mean
value of
the remaining four replicates was used. For antibodies displaying saturated
signals,
values from lower scanner settings were scaled and used instead. Chip-to-chip
normalization was performed using a semi-global normalization approach
previously
described [39, 40, 42]. First, the CV for each probe over all samples was
calculated and
ranked. Second, 15% of the probes that displayed the lowest CV-values over all
samples
were identified and used to calculate a chip-to-chip normalisation factor for
each array.
The normalization factor N, was calculated by the formula N1 = S, / p, where
Si is the sum
of the signal intensities for the antibodies used, averaged over all samples
and p is the
sample average of S,. The intensities were recalculated to log2 values prior
to statistical
analysis.
Validation of antibody specificity
The specificities of two selected scFvs (anti-IL-6 (2) and anti-IL-10 (1))
were tested using
RayBioe 278 Human Protein Array G series (Norcross, GA, USA), according to
protocol
provided by the manufacturer. The scFvs were labelled with EZ-link Sulfo-NHS-
LC-
Biotin (Pierce) for 2h on ice at a 3.5 times molar excess of biotin. Unbound
biotin was
removed by 72 h dialysis against PBS. In total, 5 pg of antibody was added to
each
array. Binding was detected using 1 pg/mL A1exa647 conjugated Streptavidin
(Invitrogen). PBS was added to one array as a negative control to check for
unspecific
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binding of Streptavidin. The arrays were scanned and the signals from the
negative
control array were subtracted. In addition, several antibody specificities
have previously
been validated using well-characterized, standardized serum samples, and
independent
methods, such as mass spectrometry, ELISA, MSD, and CBA, as well as using
spiking
and blocking experiments (Table II).
Validation of array data
A human Th1fTh2 10-plex MSD (Meso Scale Discovery, Gaithersburg, MD, USA)
assay
was run in an attempt to validate the antibody microarray results. Each well
of the MSD
96-plate had been pre-functionalized with antibodies against IFN-y, IL-16, IL-
2, IL-4, IL-5,
IL-8, IL-10, IL-12p70, IL-13 and INF-a in spatially distinct electrode spots.
A total of 34
serum samples (undiluted) were analyzed, including 11 PaC, 11 healthy, 9 ChP
and 3
AIP samples (the low number of AIP samples was due to limited sample volumes
in that
subgroup). The assay was run according to the protocol provided by the
manufacturer
and the electrochemiluminiscence-based readout was performed in an MSD SECTOR
instrument.
Microarray data analysis
All statistics and data analysis was performed in R (http://www.r-
project.org). Briefly, a
support vector machine (SVM) was employed to classify the samples as belonging
to
one of two defined groups (e.g. cancer or healthy), using a linear kernel with
the cost of
constraints set to I. No attempts were made to tune it in order to avoid the
risk of over-
fitting. The SVM was trained and tested using a leave-one-out (L00) cross
validation
procedure [42]. In two of the comparisons, this training part included
creating an antibody
sub-panel by selecting antibodies that, in the training set, displayed the
highest
discriminatory power. This selection of antibodies was made using either a
direct or a
cross-validated backward elimination strategy. Using this approach, condensed
candidate biomarker signatures were identified, and subsequently evaluated on
independent test sets.
Sensitivity and specificity values were calculated from the SVM decision
values, using a
threshold level of zero. A receiver operating characteristics (ROC) curve was
constructed
using the SVM decision values. The area under the curve (AUC) was calculated
and
used as a measure of prediction performance. Further, the Wilcoxon p-value and
the fold
change were calculated for each antibody. The candidate biomarker signatures
were
reported following the recommendations for tumour marker prognostic studies
[47].
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Biomarker signatures identification
A backward elimination procedure was used for identifying a biomarker
signature for
distinguishing PaC from healthy individuals. In this approach, one sample at
the time was
excluded from the dataset. The remaining samples were used for training the
SVM by
excluding one antibody at the time and performing the classification using the
remaining
antibodies. When all antibodies had been left out once, the least informative
antibody
was defined as the one that had been excluded when the smallest Kullback-
Leibler (KL)
error was obtained for the classification, and was eliminated from the
dataset. The LOO
procedure was iterated until only one antibody was left and the order by which
the
antibodies had been eliminated was recorded. The procedure was repeated by
excluding
a new sample and the iteration continued until all samples had been left out
once. A list
of the order in which the antibodies were eliminated was generated for each
time a
sample was excluded. In the end, all samples had been left out once and a
consensus
list was created where each antibody was assigned a score based on how long it
had
endured the elimination process, averaged over all iterations performed.
Throughout the
process, each left out sample was used to test the SVM models built for each
new length
of antibody subpanels, returning a decision value corresponding to the
performance.
Consequently, decision values for all samples for any given subpanel length
were
collected. The corresponding ROC areas were plotted against number of
antibodies as a
means to evaluate the strength of the data set and the elimination strategy. A
condensed
signature of 18 analytes was selected from the consensus list and an
independent data
set from antibody microarray analysis of 25 PaC and 20 N serum samples [39]
was used
as a test set for pre-validation of the candidate signature. The signature
analytes were
used in a SVM LOO cross validation procedure in the test set and the ability
of the
signature to distinguish PaC from N was illustrated in a ROC curve.
A second candidate biomarker signature was generated for classification of PaC
among
both N, ChP and AIP. First, the data was randomly divided into a training set
(two thirds
of the samples) and a test set (one third). A modified (even more stringent)
backward
elimination strategy was used. Instead of leaving out one sample at the time,
the SVM
was trained only once, using all samples in the training set. Consequently,
one
elimination list was generated from which a condensed panel of 25 analytes was
selected and used to build the SVM in the training set. The model was applied
onto the
independent test set and a ROC curve was generated. Furthermore, a statistical
power
analysis was performed to estimate the number of patients required in the test
set using
the function "power.t.test" in R (decision values assumed normally distributed
as
suggested by Shapiro-Wilk testing). The observed decision values from the SVM
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analysis in the training set displayed a standard deviation of 2.87 and a
delta value
between the groups of 3.47 (difference between mean values). The alpha level
(level of
significance) was set to 0.05. In addition, the validity of this backward
elimination
procedure was tested by comparing the performance of the selected signature to
1000
randomly generated signatures of the same length and to signatures generated
by
selecting the antibodies of the lowest p-value and highest fold-change,
respectively.
Finally, the strength of the classifier and the data set was tested by
comparing the
performance of the signature in the test dataset to random data, by generating
1000
permutation of the sample annotations in the test data set.
Results
Classification of PaC vs. healthy controls
In order to identify serum biomarker signature associated with PaC, we
performed
differential serum protein expression profiling of PaC (n=34) vs. N (n=30),
using the first
patient cohort. A representative image of an antibody microarray is shown in
Figure 1A,
illustrating that dynamic signal intensities, adequate spot morphology and low
non-specific background binding were obtained. The results showed that 33
non-redundant protein analytes, including e.g. both Th1 and Th2 cytokines,
were found
to be differentially expressed (p<0.05), of which all, but the complement
proteins C1q
and Properdin, were up-regulated in PaC (Fig. 1B).
To investigate whether PaC and N could be differentiated, we ran a SVM LOO
cross-validation, based on all antibodies, i.e. using unfiltered data. The
data showed that
the patient cohorts could be classified with a ROC AUC value of 0.94 (Fig.
1C). In Figure
1D, the samples are plotted by decreasing SVM decision value, and the relative
expression pattern of the top 20 differentially expressed analytes (p<0.02)
are shown in a
heat map. By using a threshold of 0 (default value), the analysis showed that
PaC vs. N
could be classified with a sensitivity and specificity of 82% and 87%,
respectively.
Next, a 278 human protein array was used for validation of selected antibody
specificities
(Figs. 1E and 1F). To this end, scFv antibodies against one highly
differentially
expressed analyte, IL-6 (p=0.005), and one modest differentially expressed
analyte,
IL-10 (p=0.04), were selected. In both cases, the protein array analysis
showed that the
scFv antibody fragments bound specifically to their target protein.
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Pre-validation of condensed biomarker signature for PaC vs. N classification
In order to test the strength of the classification derived from the first
patient cohort
(n=64), we first condensed the total number of analytes down to the 18 non-
redundant
biomarkers contributing the most to the classification, by combining our LOO
procedure
with an iterative backward elimination strategy. In this process, the Kullback-
Leibler
divergences error was minimized and used as guide for stepwise removal of the
antibodies one-by-one. After each round, the SVM decision values were
collected and
the corresponding ROC curve and AUC value were calculated. In Figure 2A, the
AUC
value is plotted against the number of remaining antibodies, indicating a high
and stable
classification even when only a few antibodies were included. The 18-analyte
condensed
candidate serum biomarker signature, composed of a variety of analytes, e.g.
cytokines,
complement proteins and enzymes, is shown in Figure 2B. Next, we applied this
18-analyte classifier on a new independent test group, the second patient
cohort (n=45)
(Fig. 2C). The results showed that the classifier allowed a stratification of
patients into
PaC vs. N with a ROC AUC value of 0.95 (Fig. 2D), corresponding to a
sensitivity of 88%
and specificity of 85%. Hence, the data outlined the first pre-validated serum
biomarker
signature for PaC diagnosis.
Biomarker signatures differentiating PaC vs. pancreatitis
To test whether cancer could be differentiated from benign conditions in the
pancreas,
we compared the serum protein expression profile of PaC (n=34) with that of
ChP (n=16)
or AIP (n=23) using the first patient cohort. In the case of PaC vs. ChP, 15
non-redundant differentially expressed (p<0.05) serum analytes were pin-
pointed, of
which all but two (IL-4 and IL-12) were up-regulated in PaC (Fig. 3A). Based
on unfiltered
data, the results showed that PaC and ChP could be differentiated with a ROC
AUC
value of 0.86 (Fig. 3B), corresponding to a 97% sensitivity and 69%
specificity. A total of
49 non-redundant serum analytes were found to be differentially expressed in
PaC vs.
AIP, with all except for C1q and Properdin, being up-regulated in PaC (Fig.
3A). Again,
based on unfiltered data, the results showed that PaC vs. AIP could be
classified with a
ROC value of 0.99 (Fig. 3B), based on a sensitivity and specificity of 97% and
91%,
respectively.
To better reflect the clinical reality, we then investigated whether
differences could be
deciphered between PaC and the combined, heterogeneous patient group of
ChP+AIP+N, using the first patient cohort (n=103). The results showed that 47
non-redundant serum proteins were differentially expressed (p<0.05) (Fig. 3A).
A
majority of the analytes (45 of 47) were found to be up-regulated in PaC,
including a wide
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36
range of proteins. Based on unfiltered data, the results showed that PaC could
be
distinguished from this heterogeneous patient group with a ROC AUC value of
0.85 (Fig.
3B).
In an attempt to validate the array data, an independent 10-plex cytokine
sandwich
antibody microarray (MSD) was applied (Fig. 3C). However, only 1 of 10
targeted serum
analytes, IL-8, was above the lower limit of detection of the MSD assay in a
majority of
the samples. Still, the observed up-regulation of IL-8 in PaC vs. N, ChP, AIP
and
combined cohort thereof was statistically confirmed (p<0.05) by the MSD assay
in all
cases, except for PaC vs. AIP (v0.29).
Refined biomarker signature for PaC diagnosis
To test the strength of the classification of the entire first patient cohort,
including PaC, N,
ChP and AIP (n=103), we split the cohort into a training set (two thirds) and
test set (one
third) (Fig. 4A). Next, a condensed serum biomarker signature composed of the
25 non-
redundant analytes contributing the most to the classification in the training
set was
deciphered by using a direct, iterative backward elimination strategy. The 25-
analyte
condensed biomarker signature, composed of e.g. cytokines and complement
proteins,
is shown in Figure 4B. Next, we applied this 25-analyte classifier on the
independent test
set (Fig. 4C). The data showed that PaC could be pinpointed with a ROC AUC
value of
0.88 (Fig. 4C), outlining a sensitivity and specificity of 73% and 75%,
respectively.
To further challenge the classifier, we statistically evaluated its
discriminatory power.
Firstly, 1000 random signatures of the same length (25 antibodies) were
generated in the
training set and applied to the test set. The results showed that the AUC
values for the
random signatures were lower than that of the classifier biomarker signature
(AUC=0.88)
in 95% of the cases (Fig. 4D). In addition, the AUC values for the
corresponding 25-
analyte signature selected based on either lowest p-values (AUC=0.77) or
highest fold-
changes (AUC=0.78) were significantly lower than that of the classifier
signature. Hence,
the data further indicated the discriminatory power of the classifier, and the
applicability
of the backward elimination strategy for defining a condensed, high-performing
signature.
Secondly, the sample annotation of the test set was permutated 1000 times in
order to
compare the specific classification to random classification of the same
number of
samples. The results showed that a significantly higher AUC value (0.88 vs.
0.19-0.86,
median value of 0.5) was obtained when the correct sample annotation was used
than
when the random annotation was applied, further demonstrating the strength of
the
classification.
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Discussion
In this study, we have applied affinity proteomics in order to harness the
diagnostic
power of the immune system to target PaC. We based our approach on the notion
that
the immune system is exquisitely sensitive to alterations in an individual's
state of health,
resulting from disease, registering these changes through fluctuations in the
levels of, in
particular, immunoregulatory analytes. To this end, we designed our antibody
microarray
to target predominantly these kinds of key regulatory serum analytes. The data
showed
that PaC-associated candidate biomarkers signatures displaying high diagnostic
power
could be de-convoluted. In a similar fashion, this affinity proteomic approach
has recently
allowed the identification of several serological biomarker signatures
distinguishing other
cancer indications and healthy controls [14-15, 17, 19], further demonstrating
the
strength of the platform.
We showed for the first time that serum stored information enabling us to
discriminate
between not only well-defined patient cohorts of PaC vs. controls and PaC vs.
pancreatitis, but also between PaC vs. the combined cohort of controls and
pancreatitis
patients with high confidence. This latter finding was in particular critical,
since the
candidate biomarker signatures must perform well also in clinical settings
where
heterogeneous patients groups will be screened.
The clinical impact of a high-performing PaC classifier would be high as no
validated
serological discriminator is yet in place [2, 7-9, 20-21]. While waiting for a
golden
classifier to be established, CA-19-9 remains the most useful molecular marker
for PaC
diagnosis [2, 8-10]. Notably, our data showed a significantly higher median
sensitivity
(88%) and specificity (85%) for PaC diagnosis than what have been consistently
observed for CA-19-9 [2, 9-11], outlining a significant clinically added
value. Further, we
have recently modelled the impact of new diagnostic possibilities on cost,
survival, and
quality of life for risk patients, and showed that affinity proteomics had
great prospects for
becoming a cost-effective tool in screening for PaC (Bolin et al, ms in
prep.).
The classifier will perform at its best if early diagnosis, when the tumour is
still small and
operable, could be performed [2, 9].
In the quest for cancer biomarkers, systemic inflammation is frequently
highlighted as a
potential confounding factor [23], since cancer development and inflammation
has been
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linked. In early works based on affinity proteomics, the results also often
showed that
general disease (inflammatory) signatures rather than cancer-specific
fingerprints were
delineated [24-26]. Notably, we showed here that PaC and pancreatitis could be
discriminated with high confidence. Furthermore, the observed signature(s)
showed
significant differences, i.e. only small overlaps, with those observed for
other various
inflammatory conditions refs [19, 27] (Carlsson etal., ms in prep.) and other
cancers [14-
15, 17, 19], further supporting the notion that PaC-specific signatures were
deciphered.
The serum immunosignatures could be considered as snapshots of the immune
system's
activity in a patient at the time of the test. These fingerprints will reflect
a combination of
direct and indirect (systemic) effects in response to the cancer. Focusing on
the cytokine
expression profiles, previous reports have shown that pancreatic cancer cell
lines
expressed a set of cytokines found to be over expressed also in this study,
including e.g.
IL-6, IL-8, IL-10, IL-12, IL-13, IL-18, and TGF-131 [28]. Several of these and
other
cytokines (e.g. VEGF and IL-7) have also been found to be overexpressed in PaC
tumour tissue and/or serum/plasma [29-33] further supporting our observations.
Although
cytokines play a pivotal role in the immune system, interpreting these
intricate expression
patterns in a biological context is demanding since many of these analytes
display
pleiotropic functions and PaC is characterized by peculiar cytokine expression
patterns
[29]. While the expression of e.g. IFN-y could signal an attempted anti-tumour
immune
response [29], the immunological environment of PaC has often been found to be
in an
immunosuppressive site, as illustrated by the concomitant expression of anti-
inflammatory cytokines (e.g. TGF-I3 and IL-10), and potentially inactive
proinflammatory
cytokines (e.g. IL-12 and IL-18) [29]. A cellular immunosuppression is a
striking biological
feature of PaC observed in many patients [34]. While Th2 skewed responses have
been
reported, the Th1/Th2 balance indicated here has also been observed [29, 31,
35]. The
cytokine expression pattern has also been found to reflect other parameters,
such as
survival [14, 29]. Looking at some of the non-cytokine markers, several
complements
proteins, such as C3, which has been suggested to function in immune
surveillance
against tumours [36-37], and the carbohydrate antigen Lewis x have also
previously
been found to be associated with PaC [38].
Taken together, we have addressed a clinical need and demonstrated that
immunosignaturing was a powerful approach for deciphering the first pre-
validated
serological biomarker signatures for PaC diagnosis. This was achieved through
a high-
performing platform, well-controlled samples and stringent bioinformatic and
validation
approaches. The potential of the predictor signature will be further validated
in follow-up
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studies, in which independent sample cohorts will be profiled. In the end,
these findings
will provide novel opportunities for improved PaC diagnosis and thereby
enhanced
prognosis and clinical management of PaC.
References
1. Hidalgo, M., Pancreatic cancer. N Engl J Med, 2010. 362(17): p. 1605-17.
2. Chu, D., W. Koh!mann, and D.G. Adler, Identification and screening of
individuals
at increased risk for pancreatic cancer with emphasis on known environmental
and genetic factors and hereditary syndromes. JOP, 2010. 11(3): p. 203-12.
3. Jemal A, S.R., Ward E, Hao Y, Xu J, Thun MJ., Cancer statistics, 2009.
CA
Cancer J Clin, 2009. 59(4).
4. Pannela, R., et al., New-onset diabetes: a potential clue to the early
diagnosis of
pancreatic cancer. Lancet Oncol, 2009. 10(1): p. 88-95.
5. Warshaw, A.L. and C. Fernandez-del Castillo, Pancreatic carcinoma. N
Engl J
Med, 1992. 326(7): p. 455-65.
6. Galasso, D., A. Carnuccio, and A. Larghi, Pancreatic cancer: diagnosis
and
endoscopic staging. Eur Rev Med Pharmacol Sci, 2010. 14(4): p. 375-85.
7. Chen, R., et al., Proteomics studies of pancreatic cancer. Proteomics
Clin Appl,
2007. 1(12): p. 1582-1591.
8. Duffy, M.J., et al., Tumor markers in pancreatic cancer: a European
Group on
Tumor Markers (EGTM) status report. Ann Oncol, 2010. 21(3): p. 441-7.
9. Fry LC, M.K., Malfertheiner P., Molecular markers of pancreatic cancer:
development and clinical relevance. Langenbecks Arch Surg. , 2008. 393(6).
10. Koopmann J, R.C., Zhang Z, Canto MI, Brown DA, Hunter M, Yeo C, Chan
DW,
Breit SN, Goggins M., Serum markers in patients with resectable pancreatic
adenocarcinoma: macrophage inhibitory cytokine 1 versus CA19-9. Clin Cancer
Res. , 2006. 15(12).
11. Boeck S, S.P., Holdenrieder S, Wilkowski R, Heinemann V., Prognostic
and
therapeutic significance of carbohydrate antigen 19-9 as tumor marker in
patients
with pancreatic cancer. Oncology, 2006. 70(4).
12. Ingvarsson J, L.A., Sjoholm AG, Truedsson L, Jansson B, Borrebaeck CA,
Wingren C., Design of recombinant antibody microarrays for serum protein
profiling: targeting of complement proteins. J Proteome Res, 2007. 6(9).
13. Wingren C, I.J., Dexlin L, Szul D, Borrebaeck CA., Design of
recombinant
antibody microarrays for complex proteome analysis: choice of sample labeling-
tag and solid support. Proteomics, 2007. 7(17).
CA 02828338 2013-08-26
WO 2012/120288
PCT/GB2012/050483
14. lngvarsson J, W.C., Carlsson A, El!mark P, Wahren B, Engstrom G,
Harmenberg
U, Krogh M, Peterson C, Borrebaeck CA., Detection of pancreatic cancer using
antibody microarray-based serum protein profiling. Proteomics, 2008. 8(11).
15. Carlsson, A., et al., Plasma proteome profiling reveals biomarker
patterns
associated with prognosis and therapy selection in glioblastoma multiforme
patients. Proteomics Clinical Applications, 2010. 4(6-7): P. 591-602.
16. Stiderlind E, S.L., Jirholt P, Kobayashi N, Alexeiva V, Aberg AM,
Nilsson A,
Jansson B, Ohlin M, Wingren C, Danielsson L, Carlsson R, Borrebaeck CA.,
Recombining germline-derived CDR sequences for creating diverse single-
framework antibody libraries. Nat Biotechnol., 2000. 18(8).
17. Carlsson A, W.C., Ingvarsson J, Ellmark P, Baldertorp B, FernO M,
Olsson H,
Borrebaeck CA., Serum proteome profiling of metastatic breast cancer using
recombinant antibody microarrays. Eur J Cancer, 2008. 44(3).
18. McShane, L. M., et al., REporting recommendations for tumor MARKer
prognostic
studies (REMARK). Nat Clin Pract Oncol, 2005. 2(8): p. 416-22.
19. El!mark P, I.J., Carlsson A, Lundin BS, Wingren C, Borrebaeck CA.,
Identification
of protein expression signatures associated with Helicobacter pylori infection
and
gastric adenocarcinoma using recombinant antibody microarrays. Mol Cell
Proteomics., 2006. 5(9).
20. Garcea, G., et al., Molecular prognostic markers in pancreatic cancer:
a
systematic review. Eur J Cancer, 2005. 41(15): p.2213-36.
21. Rustgi, A.K., Pancreatic cancer: novel approaches to diagnosis and
therapy.
Gastroenterology, 2005. 129(4): p. 1344-7.
22. Biankin, A.V., et al., Molecular pathogenesis of precursor lesions of
pancreatic
ductal adenocarcinoma. Pathology, 2003. 35(1): P. 14-24.
23. Chechlinska, M., M. Kowalewska, and R. Nowak, Systemic inflammation as
a
confounding factor in cancer biomarker discovery and validation. Nat Rev
Cancer, 2010. 10(1): p. 2-3.
24. Orchekowski, R., et al., Antibody microarray profiling reveals
individual and
combined serum proteins associated with pancreatic cancer. Cancer Res, 2005.
65(23): p. 11193-202.
25. Gao, W.M., et al., Distinctive serum protein profiles involving
abundant proteins in
lung cancer patients based upon antibody microarray analysis. BMC Cancer,
2005.5: p. 110.
26. Miller, J.C., et al., Antibody microarray profiling of human prostate
cancer sera:
antibody screening and identification of potential biomarkers. Proteomics,
2003.
3(1): p. 56-63.
CA 02828338 2013-08-26
WO 2012/120288
PCT/GB2012/050483
41
27. Dexlin-Mellby, L., et al., Tissue proteomic profiling of preeclamptic
placenta tissue
using recombinant antibody microarrays. Proteomics - Clinical Applications,
2010.
4(10-11): p.794-807.
28. Bellone, G., et al., Cytokine expression profile in human pancreatic
carcinoma
cells and in surgical specimens: implications for survival. Cancer Immunol
Immunother, 2006. 55(6): p. 684-98.
29. Bellone G, S.C., Mauri FA, Tonel E, Carbone A, Buffolino A, Dughera L,
Robecchi A, Pirisi M, Emanuelli G., Cytokine expression profile in human
pancreatic carcinoma cells and in surgical specimens: implications for
survival.
Cancer Immunol Immunother. , 2006. 55(6).
30. Chang ST, Z.J., Horecka J, Kunz PL, Ford JM, Fisher GA, Le QT, Chang
DT, Ji
H, Koong AC., Identification of a biomarker panel using a multiplexed
proximity
ligation assay improves accuracy of pancreatic cancer diagnosis. J Transl
Med.,
2009. 7(105).
31. Poch B, L.E., Ramadani M, Gansauge S, Beger HG, Gansauge F., Systemic
immune dysfunction in pancreatic cancer patients. Langenbecks Arch Surg. ,
2007. 392(3).
32. Wigmore SJ, F.K., Sangster K, Maingay JP, Garden OJ, Ross JA., Cytokine
regulation of constitutive production of interleukin-8 and -6 by human
pancreatic
cancer cell lines and serum cytokine concentrations in patients with
pancreatic
cancer. Int J Oncol. , 2002. 21(4).
33. Frick VO, R.C., Wagner M, Graeber S, Grimm H, Kopp B, Rau BM, Schilling
MK.,
Enhanced ENA-78 and IL-8 expression in patients with malignant pancreatic
diseases. Pancreatology., 2008. 8(4-5).
34. Ungefroren, H., et al., Immunological escape mechanisms in pancreatic
carcinoma. Ann N Y Acad Sci, 1999. 880: p. 243-51.
35. Schmitz-Winnenthal FH, V.C., Zgraggen K, Galindo L, Nummer D, Ziouta Y,
Bucur M, Weitz J, Schirrmacher V, Buchler MW, Beckhove P., High frequencies
of functional tumor-reactive T cells in bone marrow and blood of pancreatic
cancer patients. Cancer Res., 2005. 65(21).
36. Chen R, P.S., Cooke K, Moyes KW, Bronner MP, Goodlett DR, Aebersold R,
Brentnall TA., Comparison of pancreas juice proteins from cancer versus
pancreatitis using quantitative proteomic analysis. Pancreas, 2007. 34(1).
37. Yu KH, R.A., Blair IA., Characterization of proteins in human
pancreatic cancer
serum using differential gel electrophoresis and tandem mass spectrometry. J
Proteome Res., 2005. 4(5).
CA 02828338 2013-08-26
WO 2012/120288
PCT/GB2012/050483
42
38. Nakano M, N.T., Ito T, Kitada T, Hijioka T, Kasahara A, Tajiri M, Wada
Y,
Taniguchi N, Miyoshi E., Site-specific analysis of N-glycans on haptoglobin in
sera of patients with pancreatic cancer: a novel approach for the development
of
tumor markers. Int J Cancer., 2008. 122(10).
39. Ingvarsson J, Wingren C, Carlsson A, et at. Detection of pancreatic
cancer using
antibody microarray-based serum protein profiling. Proteomics 2008;8.
40. Carlsson A, Wingren C, Ingvarsson J, et at. Serum proteome profiling of
metastatic breast cancer using recombinant antibody microarrays. Eur J Cancer
2008;44: 472-80.
41. Wingren C, Ingvarsson J, Dexlin L, Szul D, Borrebaeck CAK. Design of
recombinant antibody microarrays for complex proteome analysis: choice of
sample labeling-tag and solid support. Proteomics 2007;7:3055-65.
42. Carlsson A, Persson 0, Ingvarsson J, et at. Plasma proteome profiling
reveals
biomarker patterns associated with prognosis and therapy selection in
glioblastoma multiforme patients. Proteomics Clin Appl 2010;4:1-12.
43. Stiderlind E, Strandberg L, Jirholt P, et at. Recombining germline-
derived CDR
sequences for creating diverse single-framework antibody libraries. Nat
Biotechnol 2000;18:852-6.
44. Borrebaeck CAK, Wingren C. Design of high-density antibody microarrays
for
disease proteomics: key technological issues. J Proteomics 2009;72:928-35.
45. Borrebaeck CAK, Wingren C. High-throughput proteomics using antibody
microarrays: an update. Expert Rev Mol Diagn 2007;7: 673-86.
46. Ellmark P; ingvarsson J, Carlsson A, Lundin BS, Wingren C, Borrebaeck
CAK.
Identification of protein expression signatures associated with Helicobacter
pylori
infection and gastric adenocarcinoma using recombinant antibody microarrays.
Mol Cell Proteomics 2006;5:1638-46.
47. McShane LM, Altman DG, Sauerbrei W, Taube SE, Gion M, Clark GM.
REporting recommendations for tumor MARKer prognostic studies (REMARK).
Nat Clin Pract Oncol 2005;2:416-22.
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TABLE I ¨ Patient demographics of the first patient cohort
Class No. of Gender Age
patients(M/Ffunknown)Mean Range
________________________________________ (SD)
PaC 34 18/12/4 65.0 42-93
(10.4)
N 30 15/15/0 33.2 24-53
(8.6)
ChP 16 12/4/0 48.8 32-73
(14.2)
AIP 23 11/11/1 42.4 14-74
________________________________________ (18.3)
All 103 56/42/5 48.2 14-93
(18.1) _____________________________________________
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TABLE II ¨ Summary of serum biomarkers analyzed by the antibody microarrays
Antigen (no. of
clones) Antigen (no. of clones)
Angiomotin (2) IL-2 (3)
R-galactosidase (1) IL-3 (3)
Bruton tyrosine kinase IL-4 (4)**
BTK (1) IL-5 (3)**
Cl Esterase inhibitor IL-6 (4)*/**
(1) IL-7(2)
Clq (1)** IL-8 (3)**
Cls (1) IL-9(3)
C3 (2)** Integrin al (1)
C4 (1)** Integrin all (1)
C5 (2)** Leptin (1)
CD40 (4) Lewis x (2)
CD40 ligand (1) Lewis y (1)
CT-17 (control) (1) MCP-1 (3)**
Digoxin (control) (1) MCP-3 (1)
Eotaxin (3) MCP-4 (2)
Factor B (1)** Mucin-1 (6)
GLP-1 (1) Procathepsin W (1)
GLP-1 R(l) Properdin (1)**
GM-CSF (3) PSA (1)
IFN-y (2) RANTES (2)
IgM (1) Sialyl Lewis x (1)
IL-10 (3)* TGF-81 (3)
IL-11 (3) TM peptide (1)
IL-12 (4)** TNF-a (2)
IL-13 (2)** TNF-13 (4)**
IL-16 (2) Tyrosine protein kinase
IL-18(3) JAK3 (1)
IL-la (3)** VEGF (4)**
IL-18 (3)
IL-1-ra (3)
*Antibody specificity determined by protein array.
**Antibody specificity previously validated by ELISA, protein array,
blocking/spiking experiments, and/or mass spectrometry.
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TABLE III ¨ Pancreatic Cancer Diagnostic Biomarkers
Blomarker name Exemplary sequences
Interleukin-7 (IL-7) AK226000, AB102893, AB102885, P13232
Integrin a-10 Hs158237; 075578
B-galactosidase P16278
Bruton's tyrosine kinase (BTK) Q06187
Complement protein C1q (C1q) IPR001073, PR00007
Complement protein Cis (C1s) P09871
B cell receptor pEchain (IgM) e.g. P01871 (not complete protein); isotype-
specific for
IgM on Ramos B cells')
Interleukin-9 (IL-9) P15248
Integrin a-11 Q9UKX5
Janus kinase 3 protein tyrosine P52333
kinase (JAK3)
Procathepsin W P56202
Properdin P27918
TM peptide (10TM protein) NA ¨ see above
Tumour necrosis factor-a (TNF-a) P01375
Angiomotin AAG01851; Q4VCS5
Complement-1 esterase inhibitor P05155
(C1-INH)
Complement protein C3 (C3) BC150179, BC150200; P01024
Complement protein C4 (C4) BC151204, BC146673, AY379959, AL645922,
AY379927, AY379926, AY379925
Complement protein C5 (C5) BC113738, BC113740, DQ400449, AB209031, P01031
CD40 Q6P2H9
Eotaxin P51671
Complement Factor B (Factor B) P00751
Glucagon-like peptide-1 (GLP-1)
Glucagon-like peptide-1 receptor P43220
(GLP-1 R)
Granulocyte-macrophage colony- P04141
stimulating factor (GM-CSF)
Interleukin-10 (IL-10) P22301
Interleukin-11 (IL-11) P20809
Interleukin-12 (IL-12) 060595
Interleukin-13 (IL-13) P35225
Interleukin-18 (IL-18) Q14116
Interleukin-la (IL-1a) P01583
Interleukin-113 (IL-113) P01584
Interleukin-2 (IL-2) P60568
Interleukin-3 (IL-3) P08700
Interleukin-4 (IL-4) , P05112
Interleukin-5 (IL-5) BC066282, CH471062, P05113
Interleukin-6 (IL-6) P05231
Interleukin-8 (IL-8) CR623827, CR623683, DQ893727, DQ890564, P10145
Interferon-y (INF-y) P01579
Leptin P41159
Lewis X /CD15 Carbohydrate structure (not applicable)
Lewis y Carbohydrate structure (not applicable)
Monocyte chemotactic protein-1 P13500
(MCP-1)
Mucin-1 P15941
Prostate specific antigen (PSA) P07288
RANTES P13501
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Biomarker name Exemplary sequences
Sialyl Lewis x Carbohydrate structure (not applicable)
Transforming growth factor-1 P01137
(TGF-b1)
Tumour necrosis factor-6 (TNF46) P01374
Vascular endothelial growth factor P15692, P49765, P49767, =043915
(VEGF)
CD40 ligand P29965
Interleukin-16 (IL-16) Q05BE6, Q8IUU6, B5TY35
Interleukin-lra (IL-1ra) P18510
Monocyte chemotactic protein-3 BC112258, BC112260, BC092436, BC070240
(MCP-3)
Monocyte chemotactic protein-4 Q99616
(MCP-4)
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TABLE IV ¨ Pancreatic Cancer Diagnostic Biomarkers
(A) Core biomarkers
Biomarker name
Interleukin-7 (IL-7)
Integrin a-10
(B) Preferred biomarkers
Biomarker name
B-galactosidase
Bruton's tyrosine kinase (BTK)
Complement protein C1q (Gig)
Complement protein Cis (Cis)
B cell receptor pri chain (IgM)
Interleukin-9 (IL-9)
Integrin a-11
Janus kinase 3 protein tyrosine kinase (JAK3)
Procathepsin W
Properdin
TM peptide
Tumour necrosis factor-a (TNF-a)
(C)
Optional additional biomarkers
Biomarker name
Angiomotin
Complement-1 esterase inhibitor (C1-INH)
Complement protein C3 (C3)
Complement protein C4 (C4)
Complement protein C5 (C5)
CD40
Eotaxin
Complement Factor B (Factor B)
Glucagon-like peptide-1 (GLP-1)
Glucagon-like peptide-1 receptor (GLP-1 R)
Granulocyte-macrophage colony-stimulating factor (GM-CSF)
Interleukin-10 (IL-10)
Interleukin-11 (IL-11)
Interleukin-12 (IL-12)
Interleukin-13 (IL-13)
Interleukin-18 (IL-18)
Interleukin-la (IL-1a)
Interleukin-1 p (IL-113)
Interleukin-2 (IL-2)
Interleukin-3 (IL-3)
Interleukin-4 (IL-4)
Interleukin-5 (IL-5)
Interleukin-6 (IL-6)
Interleukin-8 (IL-8)
Interferon-y (IFN-y)
Leptin
Lewis X /CD15
Lewis y
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Biomarker name
Monocyte chemotactic protein-1 (MCP-1)
Mucin-1
Prostate specific antigen (PSA)
Rantes
Sialyl Lewis x
Transformign growth factor-1 (TGF-b1)
Tumour necrosis factor-13 (TNF-6)
Vascular endothelial growth factor (VEGF)
CD40 ligand
Interleukin-16 (IL-16)
Interleukin-1ra (IL-1ra)
Monocyte chemotactic protein-3 (MCP-3)
Monocyte chemotactic protein-4 (MCP-4)
TABLE V ¨ Pancreatic Cancer Diagnostic Biomarker Subsets
0
t,..)
,-,
Biomarker name PaC vs N PaC vs N+Chp+AIP PaC
vs ChP PaC vs AIP
,-,
t..)
P value Backward P value
backward P value P value =
t..)
oe
A
.
CD40 X X X
. X X
Interleukin-12 (IL-12) X X X x- I
' X X
Interleukin-3 (IL-3) X X X X -
' X X I
Interleukin-4 (IL-4) X X X -
X X
-- ----- --------- ,
Interleukin-8 (IL-8) X . X
X
õ, õ.
Monocyte chemotactic protein-1 (MCP-1) X X X ' '-
. X - X
_ -=
-
n
Mucin-1 X X X
X X ___
¨
Transforming growth factor, beta-1 (TGF-b1) X X X '
X ' X x a 0
I.)
co
Tumour necrosis factor-13 (TNF-13) X X X
X
Vascular endothelial growth factor (VEGF) X X X X
X X co
B
I\)
0
B-galactosidase X X =
1 X H
CA
o1
Bruton's tyrosine kinase (BTK) X X X ,
I X
co
CD40 ligand X X X - -
X 1
.
iv
Complement protein C1q (C1q) X X
X o,
Complement protein C3 (C3) X X X
X
Glucagon-like peptide-1 (GLP-1) X X
X
B cell receptor pLIchain (IgM) X X X X .,.
______________ X
Interleukin-10 (IL-10) X X
, X
Interleukin-11 (IL-11) X X X =
X
,
Interleukin-13 (IL-13) X X
. X IV
Interleukin-16 (IL-16) X X X X
X n
1-i
Interleukin-18 (IL-18) X X X
X
, .
Interleukin-1a (IL-1a) X X X X
X ti:1
n.)
Interleukin-1ra (IL-1ra) X X X
X c=
1--,
n.)
Interleukin-5 (IL-5) X X X
X
_
un
Interleukin-6 (IL-6) X X X
X c=
4=.
oo
c.,.)
Biomarker name PaC vs N PaC vs N+Chp+AIP PaC
vs ChP PaC vs AIP
0
____________________________________________________ P value Backward
P value ____ backward __ P value P value
,
o
Interleukin-7 (IL-7) X X X X -
X 1--,
Interferon-y (INF-y) X X X'
X
t,..)
Integrin a-11 X x" X
o
t,..)
Janus kinase 3 protein tyrosine kinase (JAK3) X. . X
X oo
oo
,`- ...--.-
-
Lewis x / CD15 X
, X
" - - ,
Procathepsin W X
X
- - =
;
Properdin X X X' X
X
Sialyl Lewis x X
, ... ,
X RITAPRIMMK
X ,
rAA,
I
C
Complement protein Cis (Cis) X
X X
Eotaxin X -.
' ' X' - - . ` X
Glucagon-like peptide-1 receptor (GLP-1 R) X
X X o
iv
Integrin a-10 X
XX co
iv
Monocyte chemotactic protein-3 (MCP-3) X - X
'` .-- X X u.)
uv,
L'i
D
i,
. __________________________________________________________________ o
Complement-1 esteras inhibitor (C1-INH) X X-5
u.)
oi
Complement protein C5 (C5) X X X X
_
co
Tumour necrosis factor-a (TNF-a) X X X X
1
_______________________________________________________________________________
_________ _ _________
I\)
E(5)
..
Interleukin-9 (IL-9) X
At, X P
F
Granulocyte-macrophage colony-stimulating factor (GM-CSF) X
X
Interleukin-2 (IL-2) X X . -
X
IV
Leptin , X ,
_______________________________________________________________________________
________________ , X n
1-i
.,
Lewis y X
X -
4")
Prostate specific antigen (PSA) . X
= X to
t,..)
Rantes X
X o
1--,
t,..)
G
7:-,..-
u,
Angiomotin X
4=.
oo
r.,.,
Biomarker name PaC vs N PaC vs N+Chp+AIP PaC
vs ChP PaC vs AIP
0
P value Backward P value
backward P value P value
Complement protein C4 (C4) X
Complement Factor B (Factor B) X
Li
oe
oe
I nterleukin-1 [3 (IL-1 (3)
X
Monocyte chemotactic protein-4 (MCP-4)
X
TM peptide i
X
co
CO
UJ
Uvi
I-,
CO
0
UJ
0
CO
oe
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TABLE VI ¨ Trained SVM program
The following parameters were obtained using the e1071 1.5-24 SVM, available
from
http://cran.r-project.org/web/packages/e1071/index.html.
(A) ¨ Definition of a condensed biomarker signature for PaC vs all ( N+Chp
+AIP)
using a backward elimination strategy
filnamn <- "PaC vs all training set.txt"
groupl <- "other"
group2 <- "PaC"
# Include
source("NaiveBayesian")
library(e1071)
# Hamta data
rawfile <- read.delim(filnamn)
# Las in grupper
groups <- rawfile[,2]
# Hamta provnamn i datafilen
samplenames <- as.character(rawfile[,1])
# Skapa dataset ur rafilen
data <- t(rawfile[,-c(1,2)])
# Log
# data <- log(data)/log(2)
# antal prover
nsamples <- ncol(data)
# Skapa antikroppsnamnlista ur NYA datafilen
ProteinNames <- read.delim(filnamn,header=FALSE)
ProteinNames <- as.character(as.matrix(ProteinNames)[1,])
ProteinNames <- ProteinNames[-(1:2)]
# Kolla antal Ab i nya datasetet
antal <- length(ProteinNames)
# Ge ratt prov- och Ab-namn
rownames(data) <- ProteinNames
colnames(data) <- samplenames
# Skapa subsets
subsetl <- is.element(groups , strsplit(groupl,",")[[1]])
subset2 <- is.element(groups , strsplit(group2,",")[[1]])
# Skapa factorlista
svmfac <- factor(rep('resti,ncol(data
)),levels=c(groupl,group2,'rest'))
svmfac[subsetl] <- groupl
svmfac[subset2] <- group2
svmfac <- svmfac[subsetlIsubset2]
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# Skapa vektor for K-L felen dar det minsta for varje signaturlangd
sparas
smallestErrorPerLength <- rep(NA,antal)
# Berakna medelvarde for varje Ab over alla prov som Or med
averages <- apply(data, 1, mean)
# Skapa vektor for Ab-ordningen efter K-L felen som erhallits nar
# respektive antikropp var satt till medelvarde.
abOrder <- rep(NA,antal)
# Skapa ett dataset att eliminera i
elimData <- data[,subsetlIsubset2]
# Lista att forvara SVM-modellerna i
models <- numeric(nsamples)
# Skapa variabel for att Mita reda pa hur mange. Ab som tagits bort
borttagna <- 0
<figref></figref><figref></figref><figref></figref><figref></figref><figref></figref><figref></figref><figref></figref><figref></figref><figref></figref><figref></figref><figref></figref><figref></figref><figref></figref><figref></figref><figref></figref><figref></figref><figref></figref>
# BEGIN BACKELIM <figref></figref><figref></figref><figref></figref><figref></figref><figref></figref><figref></figref><figref></figref><figref></figref><figref></figref><figref></figref><figref></figref><figref></figref>
<figref></figref><figref></figref><figref></figref><figref></figref><figref></figref><figref></figref><figref></figref><figref></figref><figref></figref><figref></figref><figref></figref><figref></figref><figref></figref><figref></figref><figref></figref><figref></figref><figref></figref>
print(Sys.time())
# Kor tills bara tva analyter aterstar
for(j in 1:(antal-1))
{
# Check if groups are given in correct order
control < - as.numeric(svmfac)
if(sum(control[subset1]) > sum(control[subset2]))
{
print ("ERROR: Change order of your groupl and group2!!!")
break
1
# For varje signaturlangd, dar alla Or med fran borjan, trana en
modell for
# varje N-1 kombiantion av prover med den data som finns i elimData
for (i in 1:nsamples)
{
# Modellerna sparas i en array av listor kallad models
models[i] <- list(svm(t(elimData[,-i]), svmfac[-i],
kernel="linear"))
1
# Nu Or alla modeller som behovs for LOO tranade och ska testas pa
elimData.
# I elimData satts forst en analyt till medelvarde, sen testas var och
en av
# modellerna med det pray som var borttaget nar den tranades.
# Nar alla modellerna Or testade en gang beraknas KL-fel som sparas i
errors.
# Nu satts nasta analyt till medelvarde och testprocessen gors am,
tills alla
# analyter varit medelvardeseliminerade en gang. Resultatet blir en
KL-fel
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# lista lika lang som antalet analyter som är kvar i datasetet.
# Skapa en lista med K-L fel en viss signaturlangd (antal + 1 - j
Lang)
# dar areorna for varje korning dar en Ab i taget har satts till
medelvarde
errors <- testModels(models, elimData, averages)
# Lagg namnet pa Ab med samst inverkan pa felet i abOrder
abOrder[j] <- getWorstAb(errors, row.names(elimData))
# Lagger till vardet pa det minsta felet
smallestErrorPerLength[j] <- getSmallestError(errors)
# Tar bort samsta Ab ur medelvardeslistan
averages <- getNewAverages(errors, averages)
# Tar bort samsta Ab ur elimData
elimData <- getNewElimData(errors, elimData)
# Noterar att en Ab tagits bort
borttagna <- borttagna + 1
# Ange hur manga analyter som eliminerats, samt vad klockan är.
print(paste(j, "analytes eliminated @", Sys.time()), sep=")
}
# Lagg till namnet pa sista analyetn, som aldrig blen eliminerad
abOrder[length(abOrder)] <- setdiff(ProteinNames, abOrder)
# Spara resultatet till fil
filename <- paste("Backward elimination
result(",rnorm(1)+1,").txt",sep="")
write.table(cbind(smallestErrorPerLength,abOrder), file=filename,
sep="\t", quote = F,row.names = F)
<figref></figref><figref></figref><figref></figref><figref></figref><figref></figref><figref></figref><figref></figref><figref></figref><figref></figref><figref></figref><figref></figref><figref></figref><figref></figref><figref></figref><figref></figref><figref></figref><figref></figref>
# FUNCTIONS <figref></figref><figref></figref><figref></figref><figref></figref><figref></figref><figref></figref><figref></figref><figref></figref><figref></figref><figref></figref><figref></figref><figref></figref>
<figref></figref><figref></figref><figref></figref><figref></figref><figref></figref><figref></figref><figref></figref><figref></figref><figref></figref><figref></figref><figref></figref><figref></figref><figref></figref><figref></figref><figref></figref><figref></figref><figref></figref>
# getWorstAb: Rapporterar namnet pA antikroppen som kommer tas bort
# (den dar ROC-arean var som storst)
getWorstAb <- function(errors, abNames)
{
return(abNames[order(errors, decreasing = F)[1]])
1
# testModels: testar alla modeller som finns i 'models' med alla
# analyser satta till medelvarde en gang
testModels <- function(models, elimData, averages)
{
nsamples <- ncol(elimData)
d <- as.numeric(svmfac)-1
y <- numeric(nsamples)
E <- numeric(nsamples)
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analytes <- nrow(elimData)
errors <- numeric(nrow(elimData))
for(k in 1:analytes)
{
# Satt analyt k till medelvarde i elimData
# Men spara forst analytens orginalvarde
backup <- elimData[k,]
elimData[k,] <- averages[k]
# Gor LOO loop for datasetet med de redan fardiga modellena
for (i in 1:nsamples)
{
pred <- predict(models[[i]] , t(elimData[,i]),
decision.values=TRUE)
#spara decision values
y[i] <- as.numeric(attributes(pred)$decision.values)
# Berakna "sannolikheterna"
y = 1-(1/(1 + exp(-y)))
# Berakna KL-fel nar aktuell analyt är eliminerad
for (i in 1:nsamples)
{
E[i] <- -(d[i]*log(y[i])+(l-d[i])*log(1-y[i]))
}
# Spara felet
errors[k] <- sum(E)
# Lagg tillbaka analyten
elimData[k,] <- backup
return( errors )
# getNewElimData: Valjer vilken antikropp som ska tas bort ur
tranigsdatan och tar bort den
getNewElimData <- function(errors, elimData)
{
# Positionen for det minsta felet
tasBort <- order(errors,decreasing = F)[1]
return(elimData[-tasBort,])
# getSmallestError: Rapporterar minsta K-L felet
getSmallestError <- function(errors)
return(min(errors))
# getNewAverages: skapar en ny lista med medelvarden efter att en analyt
# eliminerats.
getNewAverages <- function(errors, averages)
{
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# Positionen for det minsta felet
tasBort <- order(errors, decreasing = F)[1]
return(averages[-tasBort])
1
# getRemovedAb: tar fram ID pa analyt som eliminerats
getRemovedAb <- function(errors, abNames)
1
return(abNames[order(errors, decreasing = T)[1]])
}
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TABLE VI¨ continued
(B) ¨ Definition of a condensed biomarker signature for PaC vs N using a
modified
backward elimination strategy
# Datafil och grupper
filnamn <- "PaC_vs_N_dataset.txt"
groupl <- "N"
group2 <- "PaC"
# Las in datafil
rawfileORG <- read.delim(filnamn)
# Las in grupper
groupsORG <- rawfileORG[,2]
# Las in data
dataORG <- log(t(rawfileORG[,-c(1,2)]))
# Las in Ab-namn
ProteinNames <- read.delim(filnamn,header=FALSE)
ProteinNames <- as.character(as.matrix(ProteinNames)[1,1)
ProteinNames <- ProteinNames[- (1:2)1
# Kalla Ab ratt namn
rownames(dataORG) <- ProteinNames
# Kalla prover ratt namn
samplenamesORG <- as.character(rawfileORG[,1])
colnames(dataORG) <- samplenamesORG
# Kontro1lera antalet prover
NoSamples <- dim(rawfileORG)[1]
# Kontrollera antalet Ab
NoAntibodies <- dim(rawfileORG)[2] - 2
# Skapa subsets utifran grupepr
subsetORG1 <- is.element(groupsORG , strsplit(groupl,",")[[1]])
subsetORG2 <- is.element(groupsORG , strsplit(group2,",")[[1]])
# Skapa faktorer utifran subsets
svmfacORG <- factor(rep(lresty,ncol(dataORG
)),levels=c(groupl,group2,'rest'))
svmfacORG[subsetORG1] <- groupl
svmfacORG[subsetORG2] <- group2
# Skapa vektor och array far ROC-areor respektive Signaturer
# fran varje korning utan A provet
BestROCsForEachRun <- rep(NA,NoSamples*NoAntibodies)
dim(BestROCsForEachRun) <- c(NoSamples,NoAntibodies)
AbRemovalOrderForEachRun <- rep(NA,NoSamples*NoAntibodies)
dim(AbRemovalOrderForEachRun) <- c(NoSamples,NoAntibodies)
# Fitir varje prov i datasetet:
for(A in 1:NoSamples) # for(A in NoSamples:1)
{
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# Hamta data fran orginal-rafilen for alla prover utom A
rawfile<-rawfileORG[-A,]
# Hamta provnamn i NYA datafilen
samplenames <- as.character(rawfile[,1])
# Hamta grupper i NYA datafilen
groups <- rawfile[,2]
# Skapa dataset ur NYA datasete
runData <- t(rawfile[,-c(1,2)])
# Skapa antikroppsnamnlista ur NYA datafilen
ProteinNames <- read.delim(filnamn,header=FALSE)
ProteinNames <- as.character(as.matrix(ProteinNames)[1,])
ProteinNames <- ProteinNames[-(1:2)]
# Kolla antal Ab i nya datasetet
antal <- length(ProteinNames)
# Ge ratt prov- och Ab-namn
rownames(runData) <- ProteinNames
colnames(runData) <- samplenames
# Skapa nya subsets
subsetl <- is.element(groups , strsplit(groupl,",")[[1]])
subset2 <- is.element(groups , strsplit(group2,",")[[1]])
# Skapa fly factorlista
svmfac <- factor(rep('rest',ncol(runData
)),levels=c(groupl,group2,'rest'))
svmfac[subsetl] <- groupl
svmfac[subset2] <- group2
# Skapa vektor for ROC-areor dar den basta for varje signaturlangd
sparas
bestRocPerLength <- rep(NA,antal)
# Berakna medelvarde for varje Ab Over alla prov som Or med
averages <- apply(runData, 1, mean)
# Skapa vektor for Ab-ordningen efter ROC-areorna som erhallits nar
# respektive antikropp var satt till medelvarde.
abOrder<-rep(NA,antal)
# Skapa tranings och testset att kora
trainData <- runData
testData <- runData
# Skapa variabel for att halla reda pa hur manga Ab som tagits bort
borttagna <- 0
# Kor lika manga ganger som antalet Ab - 1
for(j in 1: (antal-l))
1
# Skapa en lista med ROC-areor en viss signaturlangd (antal + 1 - j
lang)
# dar areorna for varje kOrning dar en Ab i taget har satts till
medelvarde
ROClist <- svmForAbList(antal-borttagna, trainData, svmfac,
averages)
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# Lagg Ab med samst inverkan pa ROC-area
abOrder[j] <- getRemovedAb(ROClist,row.names(trainData))
# Skapa fly traningsdata dar samsta Ab tas bort
trainData <- getNewTrainData(ROClist, trainData)
# Tar bort samsta Ab ur medelvardeslistan
averages <- getNewAverages(ROClist, averages)
# Noterar att en Ab tagits bort
borttagna <- borttagna+1
# Lagger till vardet pa den basta ROC-arean
bestRooPerLength[j]<-getBestROC(ROClist)
# Lagg till den basta arean for aktuell langd i en lista
BestROCsForEachRun[A,] <- bestRocPerLength
# Lagg till vilken Ab som togs bort for aktuell langd i en lista
AbRemovalOrderForEachRun[A,] <- abOrder
# Skriv vilken korning som genomforts till prompten
print(paste(j, "in", A,"of", NoSamples,"at",Sys.time()))
# Include
source("NaiveBayesian")
library(e1071)
# Skapar en listamed ROC-areor for en vanda med ett antal antikroppar
# dar alla antikroppar satts till medelvarde en gang
svmForAbList <- function(abNumber, trainData, svmfac, averages)
1
testData <- trainData
ROClist <- rep(NA,abNumber)
for(k in 1:abNumber) # Byter en variabel i traningsdata till
medelvarden,
# '<Or svmloo, byter tillbaka till orginalvardena.
testData[k,] <- averages[k]
#ROClist[k] <- svmLOOvaluesBE(trainData, testData, svmfac)
ROClist[k] <- svmLOOvaluesProb(trainData, testData, svmfac)
testData[k,] <- trainData[k,]
return(ROClist)
# Rapporterar namnet pa antikroppen som kommer tas bort
# (den dar ROC-arean var som storst)
getRemovedAb <- function(ROClist, abNames)
{
return(abNames[order(ROClist, decreasing = T)[1]])
# Rapporterar storsta ROC-arean
getBestROC <- function(ROClist)
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return(max(ROClist))
1
# Valjer vilken antikropp som ska tas bort ur tranigsdatan och tar bort
den
getNewTrainData <- function(ROClist, trainData)
{
# Positionen for den storsta ROC-arean
tasBort <- order(ROClist,decreasing=T)[1]
return(trainData[-tasBort,])
}
# Valjer vilken antikropp som ska tas bort ur averages
getNewAverages <- function(ROClist, averages)
{
# Positionen for den storsta ROC-arean
tasBort <- order(ROClist,decreasing=T)[1]
return(averages[-tasBort])
}
svmLOOvaluesBE <- function(trainData, testData, svmfac)
nsamples <- ncol(trainData)
res <- numeric(nsamples)
sign <- numeric(nsamples)
for (i in 1:nsamples)
{
svmtrain <- svm(t(trainData[,-i]) , svmfac[-i] , kernel="linear" )
pred <- predict (svmtrain , t(testData[,i]) , decision.values=TRUE)
res[i] <- as.numeric(attributes(pred)$decision.values)
fcn <- colnames(attributes(pred)$decision.values)[1]
if(fcn==paste(levels(svmfac)[1],"/",levels(svmfac)[2],sep=""))1sign[i]<-
11
if(fcn==paste(levels(svmfac)[2],"/",levels(svmfac)[1],sep="")){sign[i]<-
-1}
1
res <- sign * res
ROCdata <- myROC(res,svmfac)
return(ROCdata[1])
}
# Tranar svm med viss signatur pi viss data och testar pi ett pray
testlsample <- function(dataORG, svmfacORG, A, signatureAbs)
{
svmtrain <- svm(t(dataORG[signatureAbs,-A]) , svmfacORG[-A] ,
kernel="linear")
pred <- predict(svmtrain , t(dataORG[signatureAbs,A]) ,
decision.values=TRUE)
res <- as.numeric(attributes(pred)$decision.values)
return (res)
1
svmLOOvaluesProb <- function(trainData, testData, svmfac)
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1
nsamples <- ncol(trainData)
#res <- numeric(nsamples)
#sign <- numeric(nsamples)
d <- as.numeric(svmfac)-1
y <- numeric(nsamples)
E <- numeric(nsamples)
for (i in 1:nsamples)
svmtrain <- svm(t(trainData[,-i]) , svmfac[-i] , kernel="linear")
pred <- predict(svmtrain , t(testData[,i1), decision.values=TRUE)
y[i] <- as.numeric(attributes(pred)$decision.values)
}
y = 1-(1/(1 + exp(-y)))
for (i in 1:nsamples)
{
E[i] <- -(d[i]klog(y[i])+(1-d[i])*log(1-y[i]))
1
return(l/sum(E))
}
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TABLE VI ¨ continued
(C) ¨Test of signature defined in Table 5(A)
aprk-c(
IL-3(1)
C3(1)
C5(1)
IL-7(2)
IL-4 (3)
CD40 (2)
TGF-b1 (1)
IL-12(1)
GM-CSF (1)
I/
Properdin
IgMl(E) 7
VEGF (3)
IL-16(1)
MUG-1 (P3-15)
I/ IL-la(1)
TNF-b(1)
Integrin a-10
I/ C1 est. inh.
MCP-1 (3)
MCP-3 (2)
IL-2 (3)
Eotaxin (3)
IL-11 (2)
TNF-a(1)
IL-1-ra(3)
library (MASS)
library(gplots)
library(e1071)
source("C:/Program/R/R-2.8.1/library/NaiveBayesian")
filnamn<-"PaC_all_data.txt"
rawfile <- read.delim(filnamn)
samplenames <- as.character(rawfile[,1])
groups <- rawfile[,2]
data <- t(rawfile[,-c(1,2)])
ProteinNames <- read.delim(filnamn,header=FALSE)
ProteinNames <- as.character(as.matrix(ProteinNames)[1,])
ProteinNames <- ProteinNames[-(1:2)]
rownames(data) <- ProteinNames
colnames(data) <- samplenames
group1 <- "other"
group2 <- "PaC"
nTrainingSamples <- 68
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nTestSamples <- 35
training <- data[,1:nTrainingSamples ]
test <- data[,(nTrainingSamples+1):(nTrainingSamples+nTestSamples)]
aprioriBoolean <- is.element(rownames(data) , apri)
facTr <- factor(rep("rest",ncol(training)),levels=c(groupl, group2,
"rest"))
subset1Tr <- is.element(groups[1:nTrainingSamples] , groupl)
subset2Tr <- is.element(groups[1:nTrainingSamples] , group2)
facTr[subset1Tr] <- groupl
facTr[subset2Tr] <- group2
facTe <- factor(rep("rest",ncol(test)),levels=c(groupl, group2,
"rest"))
subset1Te <-
is.element(groups[(nTrainingSamples+1):(nTrainingSamples+nTestSamples)]
, strsplit(groupl,",")[[1]])
subset2Te <-
is.element(groups[(nTrainingSamples+1):(nTrainingSamples+nTestSamples)]
, strsplit(group2,",")[[1]])
facTe[subset1Te] <- groupl
facTe[subset2Te] <- group2
svmtrain <- svm(t(training[aprioriBoolean,]) , facTr, kernel="linear")
pred <- predict(svmtrain, t(test[aprioriBoolean,]) , decision.values =
TRUE, probability = T)
res <- as.numeric(attributes(pred)$decision.values, probability = T)
facnames <- colnames(attributes(pred)$decision.values)[1]
ROCdata <- myROC(res,facTe)
ROCdata[1]
SenSpe <- SensitivitySpecificity(res,facTe)
Sensitivity <- as.numeric(SenSpe[,1])
Specificity <- as.numeric(SenSpe[,2])
omSpecificity <- 1-Specificity
plot(omSpecificity, Sensitivity, ylab="Sensitivity", xlab="1-
Specificity",type="1")
mtext(side=1, line = -1.1, paste("ROC AUC = ",signif(ROCdata[1],
digits=2)))
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TABLE VI¨ continued
(D) ¨ Final SVM model for PaC vs all (N+ChP+AIP)
$call
svm.default(x = t(training[aprioriBoolean, ]), y = facTr, kernel = "linear")
$type
[110
$kernel
[1]0
$cost
[1] 1
$degree
[113
$gamma
[1] 0.04
$coef0
[1] 0
$nu
[110.5
$epsilon
[1]0.1
$sparse
[1] FALSE
$scaled
[1] TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE
TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE
$x.scale
$x.scale$'scaled:center'
C1.est..inh. C3..1. C5..1. CD40..2.
Eotaxin..3. GM.CSF..1. IgM..B. IL.11..2.
IL.12..1. IL.16..1.
24090.45 569451.81 102936.57 22951.29 26674.95 24125.44 20855.98
14129.86 44608.14 20611.42
IL.1a..1. IL.1.ra..3. IL.2..3. IL.3..1. IL.4..3.
IL.7..2. Integrin.a.10 MCP.1..3.
MCP.3..2. MUC.1..P3.15.
219572.74 19584.88 40985.94 49070.16 24741.71 20879.60 13058.64
11227.79 14915.23 50846.38
Properdin TGF.b1..1. TNF.a..1. TNF.b..1. VEGF..3.
128296.18 22788.14 13682.89 25428.40 41955.64
$x.scale$'scaled:scale'
Ctest..inh. C3..1. C5..1. CD40..2.
Eotaxin..3. GM.CSF..1. IgM..B. IL.11..2.
IL.12..1. IL.16..1.
20404.868 122237.943 28461.795 12025.068 13215.275 16954.639 14666.366
10156.372 57988.003 13187.529
IL.1a..1. IL.1.ra..3. IL.2..3. IL.3..1. IL.4..3.
IL.7..2. Integrin.a.10 MCP.1..3.
MCP.3..2. MUC.1..P3.15.
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153112.225 11314.711 76593.575 21019.692 10105.650 19923.025 8856.321
5452.479 6368.842 28650.095
Properdin TGF.b1..1. TNF.a..1. TNF.b..1. VEGF..3.
56720.049 11069.444 8292.061 12180.614 20857.971
$y.scale
NULL
$nclasses
[112
$levels
[1] "other "PaC" "rest"
$tot.nSV
[1] 27
=
$nSV
[1] 17 10
$labels
[1] 12
$SV
C1.est..inh. C3..1. C5..1. CD40..2. Eotaxin..3.
GM.CSF..1. IgM..B. IL.11..2.
IL.12..1. IL.16..1. IL.1a..1.
Pa009 -0.75869601 -0.43683015 -0.20743017 -0.005289015 -0.38931564 0.095770897
-
0.93670642 -0.05169534 -0.30674506 -0.074417127 0.50792542
Pa038 -0.42410595 -0.73311709 -1.25244853 -0.692015029 -0.05078382 -
0.003980786
5.67933331 -0.28192859 -0.28602139 -0.214764177 0.06911333
Pa006 -0.73654816 0.24109661 -0.46040093 -1.012551914 -1.15296122 -0.841258482
-
0.72678562 -0.78781462 -0.32368921 -0.848293554 -0.69234178
Pa013 -0.21700833 1.86892400 0.58834639 -0.264721795 -0.68552957 -0.153202159 -
0.04927657 0.05757379 -0.08137510 1.221751926 1.08098684
Pa024 1.58648919 1.44742521 0.75444772 -0.284927936 -0.69406449 2.493561060 -
0.44527793 -0.39055704 0.29383416 -0.455154737 -0.81120268
Pa056 -0.36431725 -0.46909174 -0.50130198 -0.554747166 -0.35106339 -
0.494146089 -
0.17013264 -0.60888484 -0.20597864 -0.517666268 1.81952423
Pa125 -0.23467729 0.25849084 0.56420454 0.369299406 0.43426269 0.208651188
1.25308235 -0.20834865 0.08202813 -0.357037439 -0.55800518
Pa001 -0.47076992 0.56381381 0.06633327 0.435287817 0.08932416 -0.222587527 -
0.47610845 -0.27857151 -0.35763315 -0.269646986 -0.76364068
Pa010 0.05320001 -0.18419292-0.59262780 0.130515458 0.29607807 0.356063766 -
0.15926715 0.20807177 -0.15588319 0.580881124 -1.35854055
Pa021 0.38072938 -0.51120893 -0.08552594 0.833802541 0.12119111 0.162821416
0.27919151 0.38595265 -0.02303639 0.687902307 -0.40739811
Pa029 -0.39903826 1.23107814 0.03028247 -0.646633317 -0.61805906 -0.141471627 -
0.55837741 -0.62561111 -0.08591402 -0.544224442 -0.39352990
Pa048 -0.26957423 0.75584809 -0.08955982 -0.255480195 -0.87836464 -0.745964552
-
0.45255194 -0.26172904 0.03931362 -0.449749768 -1.32821555
Pa058 6.10905531 -0.04325893 0.08698841 0.870476656 0.53283461 0.979088486 -
0.13691383 -0.05059637 -0.09088942 0.270927629 -0.42939318
Pa092 -0.24149853 -0.22137826 -0.86374589 -0.004986871 -0.21952427 -
0.118794005
0.17005557 0.18556909 -0.28585022 0.326046954 -0.87098967
Pa106 -0.23439949 -0.29286642 -0.90705556 1.431821157 0.87865697 1.208446338 -
0.20106733 0.67417004 -0.14514808 1.186336278 -0.70633875
Pa128 -0.08417237 0.97358160 0.52585704 -0.062347659 -0.02788034 -0.411255681
0.46178541 -0.17013906 -0.12290537 -0.008370042 -0.89794811
Pa142 -0.50958252 -1.08085583 -1.23272636 -0.299928811 0.33070388 -0.396222532
-
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0.41999851 -0.39335783 -0.26785141 -0.089558380 -0.27466106
Pa015 -0.71635090 0.14276634 -0.74693179 -0.473760238 -1.26913415 -0.964917983
-
0.79188785 -0.58820570 -0.41838792 -0.616645822 0.34550134
Pa025 -0.67904289 1.32071592 -0.29298820 -0.497584785 -0.89584008 -0.363827564
-
0.60314141 -0.52579460 -0.42216397 -0.400049252 -0.05516486
Pa027 -0.30026876 0.52136967 -0.53568667 -0.092663002 0.38297396 0.085123350 -
0.14805742 -0.08863111 -0.32783954 -0.031648097 -0.41278449
Pa045 2.32770652 1.11229339 2.68850620 -0.053703190 -0.83048219 -0.639849777 -
0.14176235 -0.17565594 0.52874104 -0.418407376 0.50156594
Pa047 -0.07301964 -0.60084555 -1.39164969 0.347757599 -0.01329602 -0.298201129
0.07255214 0.10285325 -0.08591223 0.019651353 -0.67791456
Pal 00 -0.43294548 -0.20545382 -0.28057938 0.237411951 -0.20538311 -
0.003113888
0.42568691 -0.47951871 -0.21867907 -0.315650824 -0.68013358
Pa121 -0.05039884 1.52624676 0.54898444 -0.250353390 0.26504091 -0.228812744 -
0.10786287 -0.37683608 -0.30183383 -0.169528272 -0.60888710
Pa129 -0.04672091 -1.20455598 -0.72519120 0.144364354 0.39842641 -0.176936684
1.07960964 0.09003457 -0.28256596 0.455159702 -1.07832336
Pa137 -0.81063375 0.39213184 0.40088436 -1.227901001 -1.21269965 -0.956598086 -
0.86084278 -0.79973661 7.63985656 -0.907598547 0.72267511
Pa147 -0.13778847 0.76981229 -1.00254399-0.050061794 0.15942688 -0.300030972
0.29380087 0.02270757 -0.15355286 -0.030274049 0.78409332
IL.1.ra..3. IL.2..3. IL.3..1. IL.4..3. IL.7..2. Integrin.a.10
MCP.1..3. MCP.3..2.
MUC.1..P3.15. Properdin TGF.b1..1.
Pa009 -0.873764678 -0.411112998 -1.03076857 -0.78714053 -0.5233176752 -
1.0569348707
2.27173362 -1.25407017 -1.0252649 0.1360317 -0.2661644
Pa038 -0.206056847 0.009708982 -0.60314751 0.44373161 -0.3919296075
0.8585696202 -
0.07305799 -0.03616494 0.8055718 1.6515214 -0.1293500
Pa006 -0.961571105 -0.289056355 -0.34582654 -1.06718063 -0.6638369686 -
0.7098872057 -
1.23264385-1.31169599 -0.6500758 -1.3448447 -0.9306547
Pa013 0.234806863 2.175447589 0.01266928 0.54717541 -0.1957777100 -
0.4801514210 -
0.99759860 -0.91896270 0.6498548 -1.1706810 0.4960250
Pa024 -0.305358326 -0.221631817 0.36267874 -0.18586281 -0.2572215174 -
0.0456176001
0.08051256 0.56408243 -0.5609039 -1.1089805 -0.2477406
Pa056 0.217707838 -0.154053300 -0.72632423 -0.17209153 -0.0467640708 -
0.5423370601 -
0.53963593 -0.61602551 0.9101779 -1.0850042 -0.7841200
Pa125 0.275035954 -0.224926621 -0.11926723 1.08023147 -0.0206201972 -
0.0008422566
1.28473810 0.48915923 0.6111125 -0.3934791 0.1846749
Pa001 -0.091741993 -0.213105078 -0.47534929 -0.76802149 -0.0327458023 -
0.4608177008 -
0.98212847 -0.23474026 -0.7966689 -0.2220837 -0.4402696
Pa010 -0.464155570 -0.068647798 0.64754337 -0.22005784 -0.0766204082
0.5695475081 -
0.13335331 0.53436185 0.3457089-0.1103537 -0.2059105
Pa021 0.809674963 0.082111154 0.57592395 0.85625877 0.5975398206 0.3056060393 -
0.07617649 1.04456867 2.3882107 0.2397382 0.4214540
Pa029 -0.598511728 -0.226930137 -0.44135325 -0.88287360 -0.4211643576 -
0.5463940807 -
0.73863658 -0.13480309 -1.0338848 1.3801873 -0.9098994
Pa048 -0.540799012 -0.132230617 -0.48802315 -0.40421411 -0.3511893489 -
0.2933086328 -
0.69288196 -0.32862131 -0.5117169 -0.5428015 -0.6331645
Pa058 -0.216266096 -0.263744491 -0.02111594 0.02466775 0.7903127709 -
0.4266999225
0.04047197 0.59968815 -0.6309139 0.2644843 0.4347455
Pa092 0.188422574 -0.178937815 0.55239494 -0.03520631 0.0359698815 -
0.1928812726 -
0.60201879 -0.03639941 0.2006243 -0.6324198 -0.2174438
Pa106 0.134678555 -0.023059591 0.13626501 0.32218390 -0.0635055923 -
0.0806119478
0.09892621 0.72394826 -0.4466987 0.3305753 0.8539264
Pa128 0.007572306 -0.139367554 0.21470674 -0.12000217 -0.1506709444
0.2875368605 -
0.35548071 0.63819849 -0.4067314 1.0337810 0.1537436
Pa142 -0.266469132 -0.281152595 0.82774995 -0.25295292 -0.3165063500 -
0.3388735672
1.71680502 0.49242804 -0.2902914 1.5769876-0.3204796
Pa015 -0.414827646 -0.335150876 -0.59577082 -0.84721879 -0.6165233284 -
0.5728379380 -
1.06685693 -1.32707320 -0.8710250 -1.3049299 -0.7987132
Pa025 -0.614322842 -0.351470829 -0.93514922 -1.21928380 -0.5784378396 -
0.7578714150 -
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1.07065042 -0.36768309 -1.0996868 -1.1292653 -0.8399010
Pa027 -0.289995023 -0.234718460 -0.34640819 -0.11023567 0.0001702044
0.0004392925 -
0.35285696 -0.23601985 0.5504571 -0.8609139 0.5920421
Pa045 -0.071721708 -0.122541540 1.74977696 0.33065121 -0.3344757232 -
0.0134402551
0.14558436 -0.40419305 0.5895674 -0.9058004 -0.2084024
Pa047 0.579454777 -0.023082065 1.00137061 -0.02504929 0.3732057511
0.4029526915
0.28311658 0.20414137 -0.3904702 1.3038092 -0.3220880
Pa100 -0.409728805 -0.323942678 0.13563267 -0.32632146 0.2183483192 -
0.5858327012 -
0.32588781 0.36754365 -0.8761754 1.0424584 -0.3768059
Pa121 -0.177281870 -0.189628137 0.04549011 -0.22387032 0.0960551709 -
0.1245495609 -
0.01148733 0.08500916 -0.2435519 -0.4560824 -0.2646761
Pa129 0.211284366 -0.255781747 0.20501384 -0.08248423 0.7175913867
0.0496293721
0.39857908 0.87914844 0.9371010 -0.3958375 0.6287985
Pa137 -0.641370977 -0.393683315 -1.25425888 1.23623466 -0.7024639259 -
0.6739023588 -
0.83664778 -0.50259826 -1.1584413 -1.1886815 -1.1372398
Pa147 0.091538371 -0.178739738 0.06208210 0.04644500-0.0264655443 0.1878050701
0.50826277 -0.60143916 1.5374629 1.3112835 0.0817073
TNF.a..1. TNF.b..1. VEGF..3.
Pa009 0.40813630 -0.29072629 0.21376386
Pa038 -0.18826712 0.01047747 -0.29116552
Pa006 -0.66615076 -1.03733849 -0.95344856
Pa013 0.13709356 -0.43789203 -0.39786986
Pa024 0.35985522 -0.69484995 -0.39811383
Pa056 -0.32725609 -0.65537476 -0.81052618
Pa125 0.13099818 0.09666528 0.25745881
Pa001 -0.54730109 -0.26784028 -0.01851188
Pa010 -0.07002966 0.14109466 0.47584857
Pa021 0.35647693 0.84739885 1.41457100
Pa029 -0.70424687 0.01067955 -0.12682955
Pa048 -0.88347271 -0.37892814 0.03951195
Pa058 1.71697123 0.18848291 0.01324853
Pa092 -0.43475610 0.92880031 -0.27996250
Pa106 1.01132754 1.45346413 0.67985051
Pa128 -0.59595956 0.29156722 0.09509325
Pa142 0.12253269 0.41393499 -0.31173738
Pa015 -0.93543929 -0.62606091 -0.82759284
Pa025 -0.16053458 -0.51839963 -0.21614866
Pa027 0.20233072 0.41248908 -0.18909465
Pa045 -0.17202350 -0.50984474 0.77281999
Pa047 -0.29192335 0.22268877 0.33534050
Pa100 0.48915718 -0.30606315 -0.21250898
Pa121 -0.45976438 0.09615171 0.28373940
Pa129 0.29426203 0.62249829 0.08120801
Pa137 -0.91532270 -0.81985007 -1.12040867
Pa147 0.11963137 0.13800477 0.29478773
$index
[1] 1 3 15 16 17 21 24 26 28 30 31 32 34 37 38 42 44 47 48 50 53 54 61 64 66
67 68
$rho
[1] -0.6668827
$compprob
[1] FALSE
$probA
NULL
$probB
NULL
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$sigma
NULL
$coefs
[,1]
[1,] 0.30343126
[2,] 0.09542226
[3,] 1.00000000
[4,] 0.20660644
[5,] 0.05461905
[6,] 0.82341233
[7,] 0.50601183
[8,] 1.00000000
[9,] 0.24168457
[10,] 0.33709008
[11,] 0.15704187
[12,] 0.50405791
[13,] 0.24715788
[14,] 0.85521666
[15,] 0.20152940
[16,] 1.00000000
[17,] 0.50180868
[18,] -1.00000000
[19,] -1.00000000
[20,] -1.00000000
[21,] -0.49831606
[22,] -1.00000000
[23,] -1.00000000
[24,] -1.00000000
[25,] -1.00000000
[26,] -0.10618995
[27,] -0.43058420
$na.action
NULL
$fitted
Pa009 Pa012 Pa038 Pa042 Pa055 Pa063 Pa066 Pa069 Pa081 Pa097 Pa105 Pa108 Pal 14
Pa135 Pa006 Pa013 Pa024 Pa033 Pa039 Pa052 Pa056 Pa089 Pa096 Pa125 Pa138
other other other other other other other other other other other other other
other other other other
other other other other other other other other
Pa001 Pa007 Pa010 Pa014 Pa021 Pa029 Pa048 Pa053 Pa058 Pa088 Pa091 Pa092 Pa106
Pa110 Pa119 Pa126 Pa128 Pa133 Pa142 Pa145 Pa005 Pa015 Pa025 Pa026 Pa027
other other other other other other other other other other other other other
other other other other
other other other PaC other PaC PaC PaC
Pa034 Pa040 Pa045 Pa047 Pa070 Pa071 Pa079 Pa080 Pa085 Pa094 Pa100 Pa102 Pa113
Pa121 Pa123 Pa129 Pa137 Pa147
PaC PaC PaC PaC PaC PaC PaC PaC PaC PaC other PaC PaC PaC PaC
other PaC PaC
Levels: other PaC rest
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Table VII¨ ROC-AUC values for differentiation between (A) pancreatic cancer,
and
(B) normal, chronic pancreatitis, and/or acute inflammatory pancreatitis
ROC-AUC Biomarker signature
0.71 IL-7
0.69 Integrin a-10
0.76 IL-7 + Integrin a-10 + 1 Table IV B marker
0.79 IL-7 + Integrin a-10 + 2 Table IV B markers
0.80 IL-7 + Integrin a-10 + 3 Table IV B markers
0.79 IL-7 + Integrin a-10 + 4 Table IV B markers
0.81 IL-7 + Integrin a-10 + 5 Table IV B markers
0.81 IL-7 + Integrin a-10 + 6 Table IV B markers
0.80 IL-7 + Integrin a-10 + 7 Table IV B markers
0.84 IL-7 + Integrin a-10 + 8 Table IV B markers
0.79 IL-7 + Integrin a-10 + 9 Table IV B markers
0.80 IL-7 + Integrin a-10 + 10 Table IV B markers
0.79 IL-7 + Integrin a-10 + 11 Table IV B markers
0.76 IL-7 + Integrin a-10 + 12 Table IV B markers
The core markers + 8 preferred markers gave the best ROC-AUC value.
This signature corresponds to (core marked in red):
IL-7 + Integrin a-10 + BTK + C1q + IgM + IL-9 + Procathepsin W + properdin +
TM peptide + b-galactosidase.
However, all marker combinations had substantial predictive power.
